Use of monascus in organic acid production

ABSTRACT

The present invention provides tools and methods for producing organic acids using strains of  Monascus  which are tolerant to high organic acid concentrations at low pH.

TECHNICAL FIELD

The present invention relates to microorganisms for use in organic acidproduction and their applications.

BACKGROUND

Organic acids are widely used in food, pharmaceutical and textileindustries, and are widely recognized to be one of the key chemicalbuilding blocks in biorefining. Organic acids are also of interest forthe production of polymers which are used as biodegradable plastics.

A number of microbes are capable of producing organic acids by aerobicand anaerobic fermentation processes. Lactobacillus species arecurrently used extensively in industry for starch-based lactic acidproduction. The majority of these species lack the ability to fermentpentose sugars such as xylose and arabinose. Although Lactobacilluspentosus, Lactobacillus brevis and Lactococcus lactis are able toferment pentoses to lactic acid, pentoses are metabolized using thephosphoketolase pathway which is inefficient for lactic acid production.Indeed, in the phosphoketolase pathway, xylulose 5-phosphate is cleavedto glyceraldehyde 3-phosphate and acetyl-phosphate. With this pathway,the maximum theoretical yield of lactic acid is limited to one perpentose (0.6 g lactic acid per g xylose) due to the loss of two carbonsto acetic acid.

In most platform host organisms such as E. coli, production of organicacids at high titers is either inefficient or toxic. The production oforganic acids such as lactic acid at neutral pH typically results in theproduction of Ca-lactate, which has to be converted into lactic acid bythe addition of sulphuric acid, resulting in the formation of CaSO₄(gypsum) as by product. To produce lactic acid directly, thefermentation must be executed at low pH (preferably at least one unitlower than the pKa value of lactic acid, 3.85). Lactic acid however istoxic to microorganisms, as in its protonated form it acts as anuncoupler that destroys the membrane potential. Similar drawbacks arealso noticed when the production of other organic acids such as succinicacid and fumaric acid is increased. Thus, while quite somemicro-organisms may be tolerant to low pH only a limited number oforganisms are suitable for organic acid production in that they aretolerant to organic acids at reduced pH.

An important drawback to bacterial fermentation is the cost. As manybacteria are unable to synthesize some of the amino acids or proteinsthey need for growing and for metabolizing sugars efficiently, bacteriaoften must be fed a somewhat complex package of nutrients, increasingthe direct expense to operate the fermentation. In addition, theincreased complexity of the broth makes it more difficult to recover thefermentation product in reasonably pure form, so increased operating andcapital costs are incurred to recover the product. Also, the use of cornas the feedstock competes directly with the food and feed.

Accordingly, there remains a need for improved biocatalysts for lacticacid fermentation processes.

SUMMARY OF THE INVENTION

The present invention provides improved micro-organisms for use inorganic acid production. More particularly the invention providesmicro-organisms which are highly tolerant to high concentrations oforganic acid at low pH and thus suitable for use in organic acidproduction by genetic engineering. In further particular embodiments theinvention provides recombinant fungi, more particularly of a specieswithin the Monascus genus wherein certain exogenous and/or endogenousgenes are over-expressed and/or suppressed so that the recombinantstrains produce increased levels of organic acid when cultivated in afermentable medium. Accordingly, the recombinant Monascus strains of thepresent invention can be used for enhanced production of organic acid atlow pH thereby providing an increased supply of organic acid for use infood and industrial applications.

In a first aspect, the present invention thus provides micro-organisms,more particularly of the genus Monascus, which are tolerant to highconcentrations of organic acid at low pH. In particular embodiments theyare tolerant to increased concentrations of organic acid at a pH whichis less than 1.5 units above the pKa value of the organic acid. Moreparticularly the micro-organisms of the invention are tolerant toorganic acids such as lactic acid at a pH of less than 5, moreparticularly less than 4, even more particularly less than 3, mostparticularly less than 2.8. In further particular embodiments, they arecapable of growing in medium containing organic acid at 50 g/L, mostparticularly 100 g/L, and in particular embodiments grow in mediumcontaining organic acid of up to 150 to 175 g/L, at low pH, moreparticularly at a pH of less than 5, more particularly less than 4, evenmore particularly less than 3, most particularly less than 2.8. Inparticular embodiments of the invention, the species within the Monascusgenus is Monascus ruber. This high acid tolerance makes themparticularly suitable for use in the industrial production of organicacids, even under anaerobic or quasi-anaerobic conditions.

In particular embodiments, these micro-organisms are modified by geneticengineering to further enhance organic acid production. In particularembodiments, the micro-organisms of the invention are capable of growingon hexose and pentose sugars and convert these sugars in organic acids.In more particular embodiments the micro-organisms of the invention growon glucose and xylose at a concentration at least 50 g/L, moreparticularly at concentrations of at least 70 g/L, at least 100 g/L, atleast 200 g/L or more. Particular embodiments of the invention providethat the micro-organisms of the invention are modified by geneticengineering to enhance organic acid production, more particularly by oneor more of the following:

a) one or more recombinant genes involved in the production of theorganic acid; and/orb) one or more engineered gene deletions and/or inactivation of genesinvolved in an endogenous metabolic pathway which produces a metaboliteother than the organic acid of interest and/or wherein the endogenousmetabolic pathway consumes the organic acid of interest.

In particular embodiments, the organic acid is lactic acid, succinicacid and/or fumaric acid.

In further particular embodiments the one or more engineered genedeletions and/or inactivation of genes involved in an endogenousmetabolic pathway which produces a metabolite other than the organicacid of interest is the deletion and/or inactivation of a gene involvedin the endogenous production of ethanol. More particularly themicro-organism of the genus Monascus according to the invention is amicro-organism in which the endogenous production of pyruvatedecarboxylase is inactivated. Most particularly, the micro-organism is amicro-organism of the genus Monascus in which one or more of theendogenous PDC1, PDC2 and PDC4 genes as described herein is inactivated.In further particular embodiments, the one or more endogenousdecarboxylase coding sequences that are inactivated and/or deleted aresequences corresponding to one or more of SEQ ID NO: 3, SEQ ID NO: 4and/or SEQ NO: 5.

In certain specific embodiments, the invention provides geneticallymodified or recombinant Monascus strains that are capable of producingincreased levels of lactic acid, more particularly L-lactic acid at lowpH. More particularly, L-lactic acid is produced at a high yield fromhexose and/or pentose sugars.

In particular embodiments, the micro-organism according to the inventioncomprise a heterologous or exogenous LDH gene. Accordingly, inparticular embodiments, the invention provides genetically modified orrecombinant Monascus strains comprising a heterologous or exogenous LDHgene. In further specific embodiments, the genetically modified orrecombinant Monascus strains according to the present invention comprisea heterologous or exogenous LDH gene that encodes a functional proteinthat is at least 80% identical to a protein encoded by SEQ ID NO:1.

In certain specific embodiments, the invention provides geneticallymodified or recombinant Monascus strains that are capable of producingincreased levels of succinic acid and/or fumaric acid. In particularembodiments, the micro-organisms according to the invention comprise oneor more heterologous or exogenous genes chosen from phosphoenolpyruvate(PEP) carboxykinase gene, malate dehydrogenase gene, fumarase gene,fumarate reductase gene, isocitrate lyase gene and/or malate synthasegene. Accordingly, in particular embodiments, the invention providesgenetically modified or recombinant Monascus strains comprising one ormore genes chosen from a heterologous or exogenous phosphoenolpyruvate(PEP) carboxykinase gene, a malate dehydrogenase gene, a fumarase gene,a fumarate reductase gene a isocitrate lyase gene and/or malate synthasegene.

In certain specific embodiments, the invention provides geneticallymodified or recombinant Monascus strains that are capable of producingincreased levels of fumaric acid, which micro-organisms overexpress apyruvate decarboxylase gene, a malate dehydrogenase gene, and a fumarasegene. In particular embodiments, the Monascus strains further comprise agene encoding a dicarboxylic acid carrier for transport across themembrane. In further particular embodiments, the Monascus strainsoverexpress an endogenous pyruvate decarboxylase gene and/or malatedehydrogenase gene and optionally further express an exogenous fumarasegene and/or a dicarboxylic acid carrier gene.

The invention further envisages that the yield of organic acids, such aslactic acid, succinic acid and/or fumaric acid, can be further enhancedby inactivating endogenous Monascus genes encoding proteins involved inan endogenous metabolic pathway which produces a metabolite other thanthe organic acid of interest and/or wherein the endogenous metabolicpathway consumes the organic acid. In particular embodiments, theproduction of the metabolite other than the organic acid of interest isreduced. According to further particular embodiments, themicro-organisms according to the invention comprise at least oneengineered gene deletion and/or inactivation of an endogenous pathway inwhich the organic acid is consumed or a gene encoding a product involvedin an endogenous pathway which produces a metabolite other than theorganic acid of interest.

In more particular embodiments the micro-organisms of the strainMonascus according to the invention, are recombinant micro-organismswhich comprise one or more of the following: a recombinant gene encodinga foreign gene involved in organic acid production and/or at least oneengineered gene deletion and/or inactivation of an endogenous pathway inwhich the organic acid is consumed and/or a gene encoding an enzymeinvolved in an endogenous pathway which produces a metabolite other thanthe organic acid of interest.

In particular embodiments, the micro-organisms of the strain Monascusaccording to the invention are recombinant micro-organisms wherein saidorganic acid is lactic acid and which comprise a recombinant geneencoding Lactic acid dehydrogenase (LDH) and/or at least one engineeredgene deletion and/or inactivation of an endogenous lactic acidconsumption pathway or a gene encoding an enzyme involved in anendogenous pathway which produces a metabolite other than lactic acid.

In particular embodiments, the micro-organisms of the strain Monascusaccording to the invention are recombinant micro-organisms wherein saidorganic acid is succinic acid and/or fumaric acid and which comprise arecombinant gene encoding one or more genes encoding an enzyme of theglyoxylate cycle such as isocitrate lyase and malate synthase asdescribed above and/or at least one engineered gene deletion and/orinactivation of an endogenous succinic acid and/or fumaric acidconsumption pathway or a gene encoding a product involved in anendogenous pathway which produces a metabolite other than succinic acidand/or fumaric acid.

In particular embodiments, the invention provides genetically modifiedor recombinant Monascus strains according to the present invention thusfurther contain at least one engineered gene deletion or inactivation.In more particular embodiments, the at least one engineered genedeletion or inactivation is in a gene encoding an enzyme selected fromthe group consisting of pyruvate decarboxylase (pdc), fumaratereductase, alcohol dehydrogenase (adh), acetaldehyde dehydrogenase,phosphoenolpyruvate carboxylase (ppc), D-lactate dehydrogenase (d-ldh),L-lactate dehydrogenase (l-ldh) and any combination of said genes.

In more particular embodiments, the micro-organism is engineered toproduce lactic acid and comprises at least one engineered gene deletionand/or inactivation of an endogenous gene encoding lactatedehydrogenase. Additionally or alternatively, the miro-organism isengineered to comprise at least one engineered gene deletion orinactivation of endogenous gene encoding a cytochrome-dependent lactatedehydrogenase, such as a cytochrome B2-dependent L-lactatedehydrogenase. More particularly, the engineered gene deletion orinactivation is an inactivation of the gene comprising SEQ ID NO:2and/or SEQ ID NO: 6.

In particular embodiments of the invention provide genetically modifiedor recombinant Monascus strains according to the present inventionwherein said organic acid is succinic acid and/or fumaric acid and whichfurther contain at least one engineered gene or inactivation.

In more particular embodiments, the at least one engineered genedeletion or inactivation is in a gene encoding an enzyme selected fromthe group consisting of pyruvate decarboxylase (pdc), alcoholdehydrogenase (adh), acetaldehydedehydrogenase, phosphoenolpyruvatecarboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactatedehydrogenase (l-ldh), glycerol-3-phosphate dehydrogenase, isocitratedehydrogenase and any combination of said genes, and more particularlyat least one engineered gene deletion and/or inactivation in anendogenous gene encoding pyruvate decarboxylase (pdc), alcoholdehydrogenase (adh), isocitrate dehydrogenase and/orglycerol-3-phosphate dehydrogenase.

In particular embodiments, the invention provides genetically modifiedor recombinant Monascus strains according to the present inventionwherein said organic acid is succinic acid and which further comprise atleast one engineered gene deletion and/or inactivation in an endogenousgene encoding succinate dehydrogenase and/or fumarate reductase.

In particular embodiments, the micro-organisms according to theinvention are capable of producing the organic acid at a yield of atleast 0.5 g/L from hexose or pentose sugars or combinations of hexoseand pentose sugars. More particularly, the yield is at least 2 g/L, mostparticularly at least 5 g/L. In particular embodiments the conversionyield of consumed sugar to organic acid is at least 50%.

Particular embodiments of the invention provide genetically modified orrecombinant Monascus strains according to the present invention, whichare capable of producing lactic acid, such as L-lactic acid, succinicacid and/or fumaric acid from hexose and/or pentose sugars at a yield ofat least 2 g/L. In particular embodiments, ethanol is formed as aby-product.

In a further aspect, the present invention relates to the use of themicro-organisms provided herein in the industrial production of organicacids and/or products derived therefrom, more particularly for theindustrial production of an organic acid at a pH which is less than 1.5units above the pKa value of the organic acid. Typically this impliesproduction of an organic acid at a pH which is less than 5, moreparticularly at a pH which is about 4 or less. Indeed, as a result oftheir tolerance to organic acids at low pH the micro-organisms describedherein are particularly suitable for the high yield production oforganic acids without conversion into the ion salt. In this context,genetically modified strains capable of producing organic acids withincreased yield at low pH are of particular interest. Indeed, themicro-organisms of the present invention are capable of ensuring a highyield at limited production costs. Accordingly, in particularembodiments the invention provides high yield methods of producing acomposition comprising an organic acid comprising the steps of (i)providing a genetically modified or recombinant micro-organism accordingto the invention, and (ii) culturing said micro-organism at a pH of 2.8in the presence of a hexose or pentose sugars or combinations thereof asthe sole carbon source. The compositions obtained by the methods of thepresent invention contain high levels of lactic acid, succinic acidand/or fumaric acid at low pH without contaminating organic nutrientstypically required for the cultivation of some organisms.

In particular embodiments, methods for producing an organic acid at highyield are provided which comprise the steps of (i) providing agenetically modified or recombinant strain of a species within theMonascus genus that is tolerant to high organic acid concentration atlow pH and which has been modified to produce an organic acid at highyield from hexose or pentose sugars or combinations of hexose andpentose sugars and (ii) culturing said strain in the presence of hexoseor pentose sugars or combinations of hexose and pentose sugars. Inparticular embodiments, the methods according to the present inventionfurther comprise the step of recovering the organic acid.

In particular embodiments of the methods of the present invention, theproduced organic acid is lactic acid, succinic acid and/or fumaric acid.In more particular embodiments, the lactic acid is L-lactic acid.

In certain embodiments, the methods of producing (compositionscomprising) organic acid of the present invention result in a yield oforganic acid that is at least 2 g/L. In further particular embodiments,the titer of organic acid is between 50-100 g/L and the productivity isat least 1 g/L/hr, more particularly between 2-3 g/L/hr.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is illustrated by the following Figures which areto be considered as illustrative only and do not in any way limit thescope of the invention.

FIG. 1 illustrates an example of a transformation vector which can beused for the transformation of Monascus strains according to particularembodiments of the invention.

FIG. 2 illustrates an example of a cassette-model for vectorconstruction according to a particular embodiment of the invention.

FIG. 3 illustrates the pCGHT3 transformation vector, for use in thetransformation of Monascus strains according to particular embodimentsof the invention.

FIG. 4 illustrates the sequence of the codon optimized Bt-L-LDH gene.

FIG. 5 illustrates transformation vector pCGHTGBtLT with the codonoptimized (Bos taurus Bt) LDH gene, according to a particular embodimentof the invention.

FIG. 6 illustrate transformation vector pURGHTGBtLT with the codonoptimized (Bos taurus Bt) LDH gene based on the pUR5750 plasmidaccording to a particular embodiment of the invention.

FIG. 7 illustrates LDH activity in E. coli protein extracts afterexpression of the synthetic LDH gene, according to a particularembodiment of the invention.

FIG. 8 a illustrates glucose concentration in the media as determined byHPLC for transformants LF5-T1 and LF5-T2 and untransformed M. rubercontrol LF5 according to particular embodiments of the invention.

FIG. 8 b Lactic acid concentrations in transformants LF5-T1 and LF5-T2and untransformed M. ruber control LF5 according to particularembodiments of the invention.

FIG. 9 illustrates the L-LDH enzyme activity in the biomass of the twotransformants LF5-T1 and LF5-T2 and the untransformed M. ruber controlLF5 according to particular embodiments of the invention.

FIG. 10 illustrates the consumption of glucose by transformants and wildtypes of strains LF4, LF5 and LF6, according to particular embodimentsof the invention.

FIG. 11 illustrates the production of lactic acid by transformants andwild types of strains LF4, LF5 and LF6 according to particularembodiments of the invention.

FIG. 12 Production of ethanol by transformants and wild types of strainsLF4, LF5 and LF6. Strains were precultured in 10 ml YEPD medium for 3days.

FIG. 13 illustrates the sugar (glucose or xylose) consumption andproduct (ethanol and lactic acid) formation by transformants and wildtype strains of LF4 under aerobic and anaerobic conditions, according toparticular embodiments of the invention.

FIG. 14 illustrates the sugar (glucose or xylose) consumption andproduct (ethanol and lactic acid) formation by transformants and wildtype strains of LF5 under severely aerobic and anaerobic conditions,according to particular embodiments of the invention.

FIG. 15 illustrates the sugar (glucose or xylose) consumption andproduct (ethanol and lactic acid) formation by transformants and wildtype strains of LF6 under severely aerobic and anaerobic conditions,according to particular embodiments of the invention.

FIG. 16 illustrates the sequence of the Monascus ruber PDC1 open readingframe.

FIG. 17 illustrates the sequence of the Monascus ruber PDC2 open readingframe.

FIG. 18 illustrates an example of a transformation vector which can beused for the disruption of PDC1 of Monascus strains using geneticinselection according to particular embodiments of the invention.

FIG. 19 illustrates an example of a transformation vector which can beused for the disruption of PDC2 of Monascus strains using geneticinselection according to particular embodiments of the invention.

FIG. 20 illustrates an example of a transformation vector which can beused for the disruption of PDC2 of Monascus strains using zeocinselection according to particular embodiments of the invention.

FIG. 21 provides a schematic representation of vector pCH#PDC1 used todisrupt the PDC1 gene.

FIG. 22 provides a schematic representation of vector pCL1H#PDC1 used todisrupt the PDC1 gene and insert the LDH gene simultaneously.

FIG. 23 provides a schematic representation of vector pCN#PDC2 used todisrupt the PDC2 gene in a PDC1-LDH+transformant.

FIG. 24 illustrates the sequence of the Monascus ruber PDC4 open readingframe (SEQ ID No: 5).

FIG. 25 illustrates Cyt-LDH activity in protein extracts from S.cerevisiae (Sc) and Monascus strains LF4, LF5, LF6, and the CBS strain,according to particular embodiments of the invention.

FIG. 26 illustrates the sequence of the Monascus ruber CYB2 open readingframe. (SEQ ID NO:2); a putative intron in the genomic sequence isindicated by small letters.

FIG. 27 illustrates the sequence of the Monascus ruber Mona00569 geneencoding cytochrome dependent L-LDH (SEQ ID No: 6).

FIG. 28 illustrates the sequence of the Monascus ruber gene encodingpyruvate carboxylase (SEQ ID NO:7)

FIG. 29 illustrates the sequence of the Monascus ruber gene encodingmalate dehydrogenase (SEQ ID NO:8, FIG. 29).

FIG. 30 provides a schematic representation of vector pCPCH#PDC1 used toused to disrupt the PDC1 gene and insert the endogenous pyruvatecarboxylase gene gene simultaneously.

FIG. 31 provides a schematic representation of vector pCMDHN#PDC2 usedto used to disrupt the PDC2 gene and insert the endogenous malatedehydrogenase gene.

FIG. 32 provides a schematic representation of vector pCNATFUM#PDC4 usedto disrupt the PDC4 gene and introduce the Rhizopus oryzae fumarase genecodon optimized for M. ruber.

FIG. 33 illustrates the sequence of the codon optimized Rhizopus oryzaefumarase gene.

FIG. 34 provides a schematic representation of vector pCBDAC used forthe introduction of Dicarboxylic acid carrier gene fromSchizosaccharomyces pombe codon optimized for M. ruber.

FIG. 35 illustrates the sequence of the codon optimizedSchizosaccharomyces pombe Mae-1 gene.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

As used herein, the singular forms “a”, “an”, and the include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. Where reference is madeto embodiments as comprising certain elements or steps, this impliesthat embodiments are also envisaged which consist essentially of therecited elements or steps.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of +/−10% or less, preferably +/−5% orless, more preferably +/−1% or less, and still more preferably +/−0.1%or less of and from the specified value, insofar such variations areappropriate to perform in the disclosed invention. It is to beunderstood that the value to which the modifier “about” refers is itselfalso specifically, and preferably, disclosed.

As used herein, the term “homology” denotes structural similaritybetween two macromolecules, particularly between two polypeptides orpolynucleotides, from same or different taxons, wherein said similarityis due to shared ancestry. Hence, the term “homologues” denotesso-related macromolecules having said structural similarity.

All documents cited in the present specification are hereby incorporatedby reference in their entirety.

As used herein, sequence identity between two polypeptides can bedetermined by optimally aligning (optimal alignment of two proteinsequences is the alignment that maximises the sum of pair-scores lessany penalty for introduced gaps; and may be preferably conducted bycomputerised implementations of algorithms, such as “Clustal” W usingthe alignment method of Wilbur and Lipman, 1983 (Proc. Natl. Acad. Sci.USA, 80: 726-730). Alternative methods include “Gap” using the algorithmof Needleman and Wunsch 1970 (J Mol Biol 48: 443-453), or “Bestfit”,using the algorithm of Smith and Waterman 1981 (J Mol Biol 147:195-197), as available in, e.g., the GCG™ v. 11.1.2 package fromAccelrys) the amino acid sequences of the polypeptides and scoring, onone hand, the number of positions in the alignment at which thepolypeptides contain the same amino acid residue and, on the other hand,the number of positions in the alignment at which the two polypeptidesdiffer in their sequence. The two polypeptides differ in their sequenceat a given position in the alignment when the polypeptides containdifferent amino acid residues at that position (amino acidsubstitution), or when one of the polypeptides contains an amino acidresidue at that position while the other one does not or vice versa(amino acid insertion or deletion). Sequence identity is calculated asthe proportion (percentage) of positions in the alignment at which thepolypeptides contain the same amino acid residue versus the total numberof positions in the alignment. In particular embodiment the algorithmfor performing sequence alignments and determination of sequenceidentity is one based on the Basic Local Alignment Search Tool (BLAST)originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10),more particularly the “Blast 2 sequences” algorithm described byTatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), such asusing defaults settings thereof.

As used herein, “sequence similarity” between two polypeptides can bedetermined by optimally aligning (see above) the amino acid sequences ofthe polypeptides and scoring, on one hand, the number of positions inthe alignment at which the polypeptides contain the same or similar(i.e., conservatively substituted) amino acid residue and, on the otherhand, the number of positions in the alignment at which the twopolypeptides otherwise differ in their sequence. The two polypeptidesotherwise differ in their sequence at a given position in the alignmentwhen the polypeptides contain non-conservative amino acid residues atthat position, or when one of the polypeptides contains an amino acidresidue at that position while the other one does not or vice versa(amino acid insertion or deletion). Sequence similarity is calculated asthe proportion (percentage) of positions in the alignment at which thepolypeptides contain the same or similar amino acid residue versus thetotal number of positions in the alignment.

The term “organic acid” as used herein refers to an organic compoundwith acidic properties. More particularly in the context of the presentinvention organic acid compounds are selected from the group consistingof lactic acid (2-hydroxypropionic acid), succinic acid,furandicarboxylic acid, fumaric acid, maleic acid, citric acid, glutamicacid, aspartic acid, acrylic acid, oxalic acid, and glucanic acid. Moreparticularly, in the context of the present invention organic acidcompounds are selected from the group consisting of lactic acid,succinic acid and/or fumaric acid.

The term “carboxylic acid” refers to an organic acids characterized bythe presence of at least one carboxyl group. Acids with two or morecarboxyl groups are also referred to as dicarboxylic, tricarboxylic,etc. Examples of carboxylic acids include but are not limited to oxalicacid and mal(e)ic acid, and succinic acid.

The term “lactic acid” in this application refers to 2-hydroxypropionicacid in either the free acid or salt form. The salt form of lactic acidis referred to as “lactate” regardless of the neutralizing agent, i.e.calcium carbonate or ammonium hydroxide. As referred to herein, lacticacid can refer to either stereoisomeric form of lactic acid (L-lacticacid or D-lactic acid). The term lactate can refer to eitherstereoisomeric form of lactate (L-lactate or D-lactate). When referringto lactic acid production this includes the production of either asingle stereoisomer of lactic acid or lactate or a mixture of bothstereoisomers of lactic acid or lactate. In the particular embodimentswhere the recombinant fungi of the present invention exclusively producea single stereoisomer of lactic acid or lactate, the lactic acid orlactate stereoisomer that is produced is said to be “chirally pure”. Thephrase “chirally pure” indicates that there is no detectablecontamination of one stereoisomeric form of lactic acid or lactate withthe other stereoisomeric form (the chiral purity of the specifiedstereoisomer is at least, greater than (or greater than or equal to)99.9%).

The term “succinic acid” in this application refers to butanedioic acidin either the free acid or salt form. The salt form of succinic acid isreferred to as “succinate” regardless of the neutralizing agent. Theterm “fumaric acid” in this application refers to trans-butenedioic acidin either the free acid or salt form. The salt form of fumaric acid isreferred to as “fumarate” regardless of the neutralizing agent.

The organism according to the present invention have been found to behighly tolerant to organic acids at low pH.

As used herein the terms “tolerance to high organic acid concentration”and more particularly “tolerance to high lactic acid, succinic acidand/or fumaric acid concentration” refers to the ability of the microorganisms to grow in a medium comprising at least 50 g/L organic acid,or at least 75 g/L but potentially up to more than 100 g/L, or even morethan 150 g/L, such as 175 g/L or 200 g/L. In particular embodiments, theterm “high organic acid concentration”, may refer to a saturatedsolution of the organic acid.

As used herein the term “low pH”, refers to a pH of between 2.0 and 5.0,such as less than 4.0, more particularly less than 3.0, moreparticularly a pH of 2.8 or less.

It will be understood to the skilled person that when referring to atolerance to an organic acid at a low pH, of particular relevance is theability to tolerate an organic acid at a pH which corresponds to or islower than the pKa value of the organic acid, more particularly at a pHwhich is between 1.5 unit higher and 1.5 unit lower than the pKa valueof the organic acid. Where the organic acid has two pKa values (due tothe presence of two acid groups), the relevant pKa is the lowest pKavalue.

“Lactate dehydrogenase activity” as used herein refers to the ability ofthe protein to catalyze the reaction of pyruvate to lactate. Lactatedehydrogenase enzymes include (but are not limited to) the enzymescategorized by the Enzyme Commission numbers EC1.1.1.27 and EC1.1.1.28.

The terms “recombinant” or “genetically modified” as used herein withreference to host organisms, microorganisms or cells, encompass suchhost organisms, microorganisms or cells into which a nucleic acidmolecule has been introduced or which has been in another waygenetically modified, as well as the recombinant progeny of such hostorganisms, microorganism or cells. This includes both organisms in whichendogenous gene sequences are introduced at a position other than theirnatural position in the genome and organisms in which endogenous genesequences have been modified or deleted. The term “recombinant” as usedherein with reference to a nucleotide sequence present in a hostorganisms, microorganism or cell refers to a nucleotide sequence whichis not naturally present in said organisms, microorganism or cell. Thisincludes nucleotide sequences which are foreign to said organisms,microorganism or cell and nucleotide sequences which are introduced at aposition other than their natural position in the genome and endogenousgene sequences have been modified. In particular embodiments thereference to the presence of a recombinant gene implies over-expressionof said gene in the host, i.e. increase expression of said gene in thehost as a result of genetic modification of said host.

The term “transformation” encompasses the introduction or transfer of aforeign nucleic acid such as a recombinant nucleic acid into a hostorganism, microorganism or cell. The so-introduced nucleic acid or theresulting deletion of endogenous nucleic acid is preferably maintainedthroughout the further growth and cell division of said host organism,microorganism or cell. Any conventional gene transfer or geneticmodification methods may be used to achieve transformation, such aswithout limitation electroporation, electropermeation, chemicaltransformation, lipofection, virus- or bacteriophage-mediatedtransfection, etc.

The term “gene” as generally used herein refers to a sequence whichcontains a coding sequence a promoter and any other regulatory regionsrequired for expression in a host cell.

As used herein, the term “promoter” refers to an untranslated sequencelocated within 50 bp upstream the transcription start site and whichcontrols the start of transcription of the structural gene. Generally itis located within about 1 to 1000 bp, preferably 1-500 bp, especially1-100 bp upstream (i.e., 5′) to the translation start codon of astructural gene. Similarly, the term “terminator” refers to anuntranslated sequence located downstream (i.e., 3′) to the translationstop codon of a structural gene (generally within about 1 to 1000 bp,more typically 1-500 base pairs and especially 1-100 base pairs) andwhich controls the end of transcription of the structural gene. Apromoter or terminator is “operatively linked” to a structural gene ifits position in the genome relative to that of the structural gene issuch that the promoter or terminator, as the case may be, performs itstranscriptional control function.

As used herein, the term “heterologous” or “exogenous” refers to thefact that the gene or coding sequence under consideration is not nativeor endogenous to the host.

The term “native” or “endogenous” is used herein with respect to geneticmaterials (e.g., a gene, promoter or terminator) that are found (apartfrom individual-to-individual mutations which do not affect function)within the genome of wild-type cells of the host strain.

By “encoding” is meant that a nucleic acid sequence or part(s) thereofcorresponds, by virtue of the genetic code of an organism in question,to a particular amino acid sequence, e.g., the amino acid sequence of adesired polypeptide or protein. By means of example, nucleic acids“encoding” a particular polypeptide or protein may encompass genomic,hnRNA, pre-mRNA, mRNA, cDNA, recombinant or synthetic nucleic acids.

Preferably, a nucleic acid encoding a particular polypeptide or proteinmay comprise an open reading frame (ORF) encoding said polypeptide orprotein. An “open reading frame” or “ORF” refers to a succession ofcoding nucleotide triplets (codons) starting with a translationinitiation codon and closing with a translation termination codon knownper se, and not containing any internal in-frame translation terminationcodon, and potentially capable of encoding a polypeptide. Hence, theterm may be synonymous with “coding sequence” as used in the art.

The micro-organisms of the order of Monascus according to the presentinvention, have an excellent tolerance to high organic acidconcentrations at low pH. More particularly, they are tolerant toorganic acids at a pH which is less than 1.5 units higher than the(lowest) pKa value of the organic acid. In particular embodiments, themicro-organisms are tolerant to all organic acids. Most particularlythey are tolerant to high concentrations of carboxylic organic acids atlow pH.

The tolerance to organic acids of the micro-organisms according to thepresent invention makes them particularly suitable for use in theindustrial production of organic acids such as, but not limited tolactic acid, succinic acid and/or fumaric acid. While in practice in anindustrial setting the organic acid of interest may be extracted duringproduction such that extremely high concentrations are not maintained,the tolerance of the strain to the organic acid will nevertheless beadvantageous. More particularly, it is of interest to be able tomaintain the strain at a pH value which is less than 1.5 above the(lowest) pKa value of the organic acid of interest, most particularly,where the micro-organism is tolerant to the organic acid at a pH whichis less than the (lowest) pKa value of the organic acid of interest. ThepKa values of organic acids vary between about 3.5 and 6. A non-limitinglist of exemplary organic acids, their pKa values and the correspondingpH range at which the organisms are preferably grown for industrialproduction of the organic acid is provided in Table 1.

TABLE 1 exemplary organic acids Organic acid pKa pH range Lactic acid3.85 2.5-5   Malic acid  3.4-5.11 2.0-4.5 Succinic acid 4.16-5.663.0-5.5 Acrylic acid 4.25   3-5.5 Fumaric acid 3.03-4.44 2.0-4.5 Citricacid 3, 13-4.76, 6.4 2.0-4.5

The organisms according to the present invention have been found to betolerant to a range of organic acids, at low pH. In particularembodiments, they are tolerant to carboxylic acids, at a pH which islower than 1.5 units above the (lowest) pKa value of the organic acid.In particular embodiments, the tolerance to organic acids which do notcontain a double bonded CH₂ group is increased. In further particularembodiments, the micro-organisms are tolerant to organic acids at a pHwhich is lower than the pKa value of the relevant organic acid. Moreparticularly, the micro-organisms of the present invention have anexceptional tolerance to lactic acid, succinic acid and/or fumaric acidat a pH of less than 3.

It has moreover been found that the organisms according to the presentinvention can be engineered to ensure production of an organic acid.More particularly they can be engineered to ferment simple sugars to anorganic acid at high yield. The fact that in particular embodimentsthese organisms can be cultivated under anaerobic or quasi-anaerobicconditions, provides an additional advantage in the context ofindustrial production methods.

The micro-organisms according to the invention are of a species withinthe Monascus genus. It has surprisingly been found that Monascus strainscan be identified which are tolerant to high organic acid concentrationsat low pH. Typically the micro-organisms represent a strain of a speciesthat is chosen from the group comprising Monascus albidulus, Monascusargentinensis, Monascus aurantiacus, Monascus barkeri, Monascusbisporus, Monascus eremophilus, Monascus floridanus, Monascusfuliginosus, Monascus fumeus, Monascus kaoliang, Monascus lunisporas,Monascus mucoroides, Monascus olei, Monascus pallens, Monascus paxii,Monascus pilosus, Monascus pubigerus, Monascus purpureus, Monascusruber, Monascus rubropunctatus, Monascus rutilus, Monascus sanguineus,Monascus serorubescens and Monascus vitreus. In particular embodimentsof the present invention, the micro-organisms of the present inventionare strains of the species Monascus ruber.

Exemplary strains according to the present invention, referred to hereinalso as LF4, LF5 and LF6, have been deposited at the Centraalbureau voorSchimmelcultures (CBS) in Utrecht, The Netherlands and have beenattributed the accession numbers CBS 127564, CBS 127565 and CBS 127566,respectively.

In particular embodiments, the invention relates to micro-organisms ofthe order of Monascus as described above, which have been geneticallyengineered to improve organic acid yield, more particularly theproduction of an organic acid, such as lactic acid, succinic acid and/orfumaric acid from simple sugars such as hexose or pentose sugars orcombinations of hexose and pentose sugars. This can be achieved indifferent ways, which are not mutually exclusive.

In particular embodiments, the genetic engineering techniques envisagedin the context of the present invention aim at the increase organic acidproduction by expression of an (exogenous) gene encoding an enzymeinvolved in the production of the organic acid of interest.

In particular embodiments, the organic acid of interest is lactic acid.More particularly, overexpression of an (exogenous) lactatedehydrogenase gene is envisaged. Lactate dehydrogenase catalyzes thelast step in the conversion of sugars to lactic acid, whereby pyruvateis converted to lactate. Increased expression and/or activity of LDHgenes thus ensures an increase in lactic acid yield. Accordingly, inparticular embodiments, the invention provides genetically modified orrecombinant Monascus strains comprising at least one functional lactatedehydrogenase (LDH) gene integrated into its genome. In particularembodiments, the LDH gene comprises a heterologous or exogenous LDHcoding sequence.

The introduction of an exogenous LDH gene enables the modified Monascusstrain to produce increased quantities of the L- and/or D-lactic acidstereoisomer. According to certain particular embodiments of the presentinvention, the genetically modified or recombinant micro-organisms ofthe order of Monascus comprise (or exclusively contain) one or moreexogenous L-LDH genes (and not an exogenous D-LDH gene), so that theorganisms produces an optically or chirally pure L-lactic acid.Alternatively, production of chirally pure D-lactic acid can beenvisaged by introduction of an exogenous D-LDH gene. In specificembodiments of the present invention the genetically modified orrecombinant organisms of the present invention of the order of Monascuscontain one or more exogenous L-LDH coding sequence and one or moreexogenous D-LDH coding sequences, so that the recombinant strainproduces a racemic mixture of L- and D-lactic acid.

The exogenous LDH gene used in the context of the present invention, isa gene that encodes for a lactate dehydrogenase enzyme or an activefragment thereof, i.e. a protein having lactate dehydrogenase activity.In particular embodiments, the exogenous LDH gene or coding sequence isa gene or coding sequence derived from another organism that has beengenetically modified (e.g. codons altered) for improved expression inMonascus.

In the context of the present invention, suitable LDH genes includethose obtained from bacterial, fungal, yeast or mammalian sources.Examples of specific L-LDH genes are those obtained from Lactobacillushelveticus, L. casei, Bacillus megaterium, Pediococcus acidilactici,Rhizopus oryzae and mammal sources such as bovine or swine. Inparticular Bos taurus. Examples of specific D-LDH genes are thoseobtained from L. helveticus, L. johnsonii, L. bulgaricus, L.delbrueckii, L. plantarum, L. pentosus and P. acidilactici. Functionalcoding sequences that have an identity score of at least 70% relative tothe coding sequences in these genes at the amino acid level and encodefunctional proteins are suitable. The native genes obtained from any ofthese sources may be subjected to mutagenesis if necessary to provide acoding sequence starting with the usual eukaryotic starting codon (ATG),or for other purposes. In particular embodiments, the L-LDH gene is thatobtained from L. helveticus or one that has a sequence identitytherewith of at least 80%, 85%, 90% or 95%. According to other specificembodiments, the L-LDH gene that is obtained from B. megaterium or oneat least 80%, 85%, 90% or 95% compared with such gene. According tofurther certain particular embodiments, the L-LDH gene is that obtainedfrom Bos taurus or one that has an identities score of at least 80%,85%, 90% or 95% compared with such gene. In particular embodiments, theD-LDH gene is that obtained from L. helveticus or one that has anidentity score of at least 80%, 85%, 90% more particularly at least 95%compared with such gene.

Particularly suitable LDH coding sequences include those that encode foran enzyme with an amino acid sequence that has an identity score of atleast 80%, 85% or 95%, compared with the sequence identified as SEQ. ID.NO. 1. Particularly suitable LDH genes also include those that encode afunctional enzyme having a protein sequence that has an identities scoreof at least, 80%, 85% or 95% compared to the protein sequence encoded bySEQ ID NO:1; in particular embodiments suitable LDH genes include thosethat encode a functional enzyme and having a sequence that with anidentities score of at least, 80%, 85% or 95% compared to the sequenceof SEQ ID NO:1 or identical to SEQ ID NO:1. The genetically modified orrecombinant Monascus strains of the present invention may contain asingle exogenous gene encoding an enzyme involved in the production ofthe organic acid of interest or multiple exogenous genes. Thus, forinstance, the recombinant strain may comprise a single exogenous LDHgene or multiple exogenous LDH genes, such as from 1 to 10 exogenous LDHgenes, especially from 1 to 5 exogenous LDH genes. When the transformedstrain contains multiple exogenous LDH genes, the individual genes maybe copies of the same gene, or include copies of two or more differentLDH genes. Multiple copies of the exogenous LDH gene may be integratedat a single locus (so they are adjacent to each other), or at severalloci within the genome of the host strain.

In particular embodiments, the organic acid of interest is succinic acidand/or fumaric acid. More particularly, overexpression of one or moregenes selected from an (exogenous) phosphoenolpyruvate (PEP)carboxykinase, pyruvate carboxylase, malate dehydrogenase, fumaraseand/or fumarate reductase and/or overexpression of malic enzyme isenvisaged. Phosphoenolpyruvate (PEP) carboxykinase is an enzyme in thelyase family used in the metabolic pathway of gluconeogenesis. Itconverts oxaloacetate into phosphoenolpyruvate and carbon dioxide.Whereas most reactions of gluconeogenesis can use the glycolysis enzymesin the opposite direction, the pyruvate kinase enzyme is irreversible.

According to particular embodiments of the present invention, the PEPcarboxykinase is active under anaerobic or oxygen limited conditions inthe presence of a fermentable carbon source or glycerol. A fermentablecarbon source may be glucose, fructose, galactose, raffinose, arabinose,or xylose. It was found advantageous that the Monascus comprises a PEPcarboxykinase according to the present invention, since PEPcarboxykinase catalysing the conversion from PEP to OAA fixates CO₂ andgenerates energy in the form of ATP. In a particular embodiment, theorganic acid tolerant Monascus strain according to the present inventioncomprises an enzyme having PEP carboxykinase activity, wherein theenzyme is a heterologous enzyme, preferably the heterologous enzyme isderived from a bacterium, more preferably the enzyme having PEPcarboxykinase activity is derived from Escherichia coli, Mannheimia sp.,Actinobacillus sp., or Anaeroblospirillum sp., more preferablyMannheimia succiniciproducens, Actinobacillus succinogenes, orAnaerobiospirillum succiniciproducens.

Similar to or in addition to a nucleotide sequence encoding an enzymehaving PEP carboxykinase activity, the micro-organism according to thepresent invention may be further genetically modified or transformedwith nucleotide sequences that encode homologous and/or heterologousenzymes that catalyse reactions in the micro-organism resulting in anincreased flux towards fumaric acid and/or succinic acid.

In further particular embodiments, endogenous enzymes involved in thesynthesis of succinic and/or fumaric acid are (over)expressed. Theenzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinaseprovide an alternate path to effectively reverse the actions of pyruvatekinase. Increased expression and/or activity of these genes thus ensuresan increase in succinic acid yield. More particularly, the presentinventors have identified the endogenous Monascus sequence encodingpyruvate carboxylase and malate dehydrogenase which catalyses theconversion from OAA to malic acid. Thus in particular embodiments, theMonascus strains according to the invention overexpress the pyruvatedecarboxylase and malate dehydrogenase encoded by SEQ ID NO: 7 and 8respectively.

Malic enzyme catalyzes the conversion of pyruvate to succinic acid viamalate; fumarate reductase converts fumaric acid to succinic acid;fumarase catalyses the conversion of malic acid to fumaric acid;

The introduction of one or more of the endogenous or exogenous genesdescribed above enables the modified Monascus strain to produceincreased quantities of succinic and/or fumaric acid.

Further enzymes required for the production of other organic acid areknown to the skilled person.

The recombinant coding sequence encoding the enzyme of interest isplaced under the transcriptional control of one or more promoters andone or more terminators, both of which are functional in the modifiedfungal cell.

Promoters and terminator sequences may be native to the Monascus hoststrain or exogenous to the cell. Useful promoter and terminatorsequences include those that are highly identical (i.e. have anidentities score of 90% or more, especially 95% or more, most preferably99% or more) in their functional portions compared to the functionalportions of promoter and terminator sequences, respectively, that arenative to the host strain, particularly when the insertion of theexogenous gene is targeted at a specific site in the strain's genome.

In particular embodiments the promoter has an identity score at least90%, 95% or 99% relative to a promoter that is native to a fungal gene.More particularly the promoter has an identity score of at least 90%,95% or 99% compared to a promoter for a gene that is native to theMonascus host strain. In particular embodiments, the terminator has anidentity score of at least 90%, 95% or 99% compared to a terminator fora gene that is native to a fungus. The terminator may have an identityscore of at least 90%, 95% or 99% with a terminator for a gene that isnative to the Monascus host strain. The use of native (to the hoststrain) promoters and terminators, together with their respectiveupstream and downstream flanking regions, can permit the targetedintegration of the recombinant gene into specific loci of the hoststrain's genome, and for simultaneous integration the recombinant geneand deletion of another native gene. It is possible for the differentexogenous coding sequences such as the LDH coding sequences to be placedunder the control of different types of promoters and/or terminators.

The recombinant gene may be integrated randomly into the host strain'sgenome or inserted at one or more targeted locations. Examples oftargeted locations include the loci of a gene that is desirably deletedor disrupted.

The present inventors further envisage increasing organic acidproduction in the micro-organisms of the order of Monascus according tothe invention, by limiting the production of metabolites endogenous tothe host and/or reducing endogenous consumption of the organic acid ofinterest. Indeed, this not only prevents the waste of material andenergy by the host, but pushes the host to fully rely on the productionpathway of the enzyme of interest created by introduction of therecombinant gene.

In fungi such as Monascus, lactic acid is consumed in the presence ofoxygen. This is ensured by the cytochrome dependent lactatedehydrogenase. Accordingly, additionally or alternatively, according toparticular embodiments of the present invention, the micro-organisms aremodified to reduce endogenous consumption of lactic acid. Moreparticularly, this is ensured by reducing expression of the endogenousLDH gene. This increases lactic acid yield under aerobic conditions.

Thus, according to a particular embodiment, the micro-organisms of thepresent invention have one or more inactivated endogenouscytochrome-dependent LDH gene. The present inventors have identified andcharacterized the endogenous LDH gene from Monascus ruber. In particularembodiments, the endogenous LDH comprises the coding sequence of SEQ IDNO: 2 or SEQ ID NO:6. As detailed herein below nucleotide sequencesderived from the coding, non-coding, and/or regulatory sequences of theendogenous LDH gene of Monascus ruber can be used to prevent or reduceexpression of LDH in Monascus.

In further particular embodiments, the genetic engineering techniquesused in the context of the present invention are aimed at reducing theendogenous production of metabolites other than the organic acid ofinterest. More particularly, recombinant micro-organisms are providedwhich are characterized in that enzymatic activities involved in theproduction of metabolites other than lactic acid, succinic acid and/orfumaric acid such as ethanol have been inactivated or suppressed.

In this context it is meant by “inactivate” that all or part of thecoding region of the gene is eliminated (deletion), or the gene or itspromoter and/or terminator region is modified (such as by deletion,insertion, or mutation) so that the gene no longer produces an activeenzyme, or produces an enzyme with severely reduced activity.Inactivation further includes silencing such as by antisense, triplehelix, and ribozyme approaches, all known to the skilled person.

According to particular embodiments, the genetically modified orrecombinant Monascus strains according to the present invention compriseat least one engineered gene deletion and/or inactivation, moreparticularly in an endogenous gene encoding an enzyme involved in theethanol production pathway. In these embodiments, the at least oneengineered gene deletion or inactivation can for example be in anendogenous gene encoding an enzyme that is involved in ethanolproduction pathway or in the production of other metabolites than theorganic acid of interest in the host strain, such as a gene encoding anenzyme selected from the group consisting of pyruvate decarboxylase(pdc), fumarate reductase, alcohol dehydrogenase (adh), acetylaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc), D-lactatedehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), lactate2-monooxygenase and any combination of said genes. In more particularembodiments, the at least one engineered gene deletion and/orinactivation can be in an endogenous gene encoding pyruvatedecarboxylase (pdc). Pyruvate decarboxylase catalyses the first step inthe alcohol pathway. Accordingly micro-organisms having a substantiallyreduced pyruvate decarboxylase (PDC) activity are particularlyenvisaged. The term “reduced pyruvate decarboxylase activity” meanseither a decreased concentration of enzyme in the cell (as a result ofat least one genetic modification affecting expression) and/or reducedor no specific catalytic activity of the enzyme (as a result of at leastone genetic modification affecting activity).

In particular embodiments, the invention provides micro-organisms of theorder of Monascus wherein the pyruvate decarboxylase activities approachzero or are reduced compared to the normal pyruvate decarboxylaseactivities in wild type strains. Accordingly, in particular embodimentsthe pyruvate decarboxylase activities in the strains of the presentinvention are for instance at least 60% lower, preferably at least 80%lower and even more preferably at least 90% lower than the pyruvatedecarboxylase activity detectable in of wild type strains.

According to particular embodiments of the present invention, strainsare provided, which are characterized by the feature that the ethanolproduction by said strain approaches zero or is at least reducedcompared to the background ethanol production in the wild-type strain.Accordingly, in particular embodiments, the ethanol production in thestrains of the present invention is for instance at least 60% lower,preferably at least 80% lower and even more preferably at least 90%lower than the ethanol production in the corresponding wild-type strain.

Thus, according to particular embodiments of the present invention,micro-organisms are provided wherein said organic acid is lactic acidand which comprises one or more of the following

a) a recombinant gene encoding LDH; andb) at least one engineered gene deletion and/or inactivation of anendogenous lactic acid consumption pathway or a gene encoding a productinvolved in an endogenous pathway which produces a metabolite other thanlactic acid. More particularly, said micro-organism comprises anengineered gene deletion and/or inactivation in a gene encoding anenzyme selected from the group consisting of pyruvate decarboxylase(pdc), fumarate reductase, alcohol dehydrogenase (adh),acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc),D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh) and anycombination of said genes. And more particularly, at least oneengineered gene deletion and/or inactivation in an endogenous geneencoding pyruvate decarboxylase (pdc), D-lactate dehydrogenase (d-ldh)and/or L-lactate dehydrogenase (l-ldh).

According to another particular embodiments of the present invention,micro-organisms are provided wherein said organic acid is succinic acidand/or fumaric acid and which comprises one or more of the following

a) a recombinant gene encoding phosphoenolpyruvate (PEP) carboxykinase,malate dehydrogenase, fumarase and/or fumarate reductase and/or one ormore recombinant genes encoding an enzyme of the glyoxylate cycle suchas isocitrate lyase and malate synthase; andb) at least one engineered gene deletion and/or inactivation of anendogenous succinic acid and/or fumaric acid consumption pathway or agene encoding a product involved in an endogenous pathway which producesa metabolite other than succinic acid and/or fumaric acid. Moreparticularly, said engineered gene deletion and/or inactivation in agene encoding an enzyme is selected from the group consisting ofpyruvate decarboxylase (pdc), alcohol dehydrogenase (adh),acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc),D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh),glycerol-3-phosphate dehydrogenase, Isocitrate dehydrogenase and anycombination of said genes. More particularly comprising at least oneengineered gene deletion and/or inactivation in an endogenous geneencoding pyruvate decarboxylase (pdc), alcohol dehydrogenase (adh),isocitrate dehydrogenase and/or glycerol-3-phosphate dehydrogenase.

Particularly, the micro-organism according to the present invention is amicro-organism wherein at least one gene encoding alcohol dehydrogenaseis not functional. An alcohol dehydrogenase gene that is not functionalis used herein to describe a micro-organism which comprises a reducedalcohol dehydrogenase activity compared to a micro-organism wherein allgenes encoding an alcohol dehydrogenase are functional. A gene maybecome not functional by known methods in the art, for instance bymutation, disruption, or deletion. Particularly, a micro-organism is afungi such as Monascus, wherein one or more genes adh1 and/or adh2,encoding alcohol dehydrogenase are inactivated.

Particularly, the micro-organism according to the present inventionfurther comprises at least one gene encoding glycerol-3-phosphatedehydrogenase which is not functional. A glycerol-3-phosphatedehydrogenase gene that is not functional is used herein to describe amicro-organism, which comprises a reduced glycerol-3-phosphatedehydrogenase activity, for instance by mutation, disruption, ordeletion of the gene encoding glycerol-3-phosphate dehydrogenase,resulting in a decreased formation of glycerol as compared to themicro-organism. Surprisingly, it was found that the micro-organismcomprising reduced alcohol dehydrogenase activity and/orglycerol-3-phosphate dehydrogenase activity results in an increasedproduction of succinic acid.

According to yet further particular embodiments of the presentinvention, micro-organisms are provided wherein said organic acid issuccinic acid and which comprises at least one engineered gene deletionand/or inactivation in an endogenous gene encoding succinatedehydrogenase and/or fumarate reductase. In a particular embodiment therecombinant micro-organism according to the present invention comprisesat least one gene encoding succinate dehydrogenase that is notfunctional. A succinate dehydrogenase that is not functional is usedherein to describe a micro-organism, which comprises a reduced succinatedehydrogenase activity by mutation, disruption, or deletion, of at leastone gene encoding succinate dehydrogenase resulting in a increasedformation of succinic acid as compared to the wild-type cell. Accordingto particular embodiments, the genetically modified or recombinantMonascus strains according to the present invention are further modifiedto improve consumption of pentose sugars, more particularly xyloseconsumption. This can be achieved in different ways. In particularembodiments this is achieved by introduction into the organism, anucleic acid sequence encoding xylose isomerase. Such a nucleic acid canbe from different origins, more particularly from a eukaryote, mostparticularly from a an anaerobic fungus. In a particular embodiment thexylose isomerase originates from Pyromyces sp., from a Clostridium or aFusobacterium or from Bacteroides thetaiotaomicron or Cyllamyces.Examples of suitable xylose isomerase genes are known in the art andinclude but are not limited to xylose isomerase from Piromyces sp. Whenexpressed, the sequence encoding the xylose isomerase confers toMonascus the ability to convert xylose to xylulose which may be furthermetabolised by Monascus in the production of the organic acid ofinterest. Thus, Monascus is capable of growth on xylose as carbonsource, more particularly is capable of producing the organic acid ofinterest with xylose as a carbon source, or in particular embodiments,with xylose as the only carbon source.

In further particular embodiments, the recombinant Monascus strain ismodified by introduction into the organism, a nucleic acid sequenceencoding xylose reductase and/or xylitol dehydrogenase. Such a nucleicacid can be from different origins, more particularly from a eukaryote,most particularly from Pichia stipitis. When expressed, the sequenceencoding the xylose reductase confers to Monascus the ability to producean organic acid from xylulose.

Genetic modification of the host strains is accomplished in one or moresteps via the design and construction of appropriate vectors andtransformation of the host strain with those vectors. Electroporationand/or chemical (such as calcium chloride- or lithium acetate-based)transformation methods or Agrobacterium tumefaciens-mediatedtransformation methods as known in the art can be used. The vectors caneither be cut with particular restriction enzymes or used as circularDNA. The vector used for genetic modification of the host strains may beany vector so long as it can integrate in the genome of the host strain.Vectors of the present invention can be operable as cloning vectors orexpression vectors in the selected host strain. Numerous vectors areknown to practitioners skilled in the art, and selection of anappropriate vector is a matter of choice. The vectors may, for example,be the pUR5750 transformation vector, the pCGHT3 transformation vector .. . etc.

In general, a vector is prepared that contains the coding sequence ofinterest and associated promoter and terminator sequences. The vectormay contain restriction sites of various types for linearization orfragmentation. Vectors may further contain a backbone portion (such asfor propagation in E. coli) many of which are conveniently obtained fromcommercially available yeast or bacterial vectors. The vector preferablycontains one or more selection marker gene cassettes. A “selectionmarker gene” is one that encodes a protein needed for the survivaland/or growth of the transformed cell in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins such as zeocin (sh ble genefrom Streptoalloteichus hindustanus), genetecin, melibiase (MEL5),hygromycin (aminoglycoside antibiotic resistance gene from E. coli),ampicillin, tetracycline, or kanamycin (kanamycin resistance gene ofTn903), (b) complement auxotrophic deficiencies of the cell. Twoprominent examples of auxotrophic deficiencies are the amino acidleucine deficiency (e.g. LEU2 gene) or uracil deficiency (e.g. URA3gene). Cells that are orotidine-5′-phosphate decarboxylase negative(ura3-) cannot grow on media lacking uracil. Thus a functional UBA3 genecan be used as a marker on a cell having a uracil deficiency, andsuccessful transformants can be selected on a medium lacking uracil.Only cells transformed with the functional URA3 gene are able tosynthesize uracil and grow on such medium. If the wild-type strain doesnot have a uracil deficiency (as is the case with I. orientalis, forexample.), an auxotrophic mutant having the deficiency must be made inorder to use URA3 as a selection marker for the strain. Methods foraccomplishing this are well known in the art.

Preferred selection makers include the zeocin resistance gene, G418resistance gene, hygromycin resistance gene. The selection markercassette typically further includes a promoter and terminator sequence,operatively linked to the selection marker gene, and which are operablein the host Monascus strain.

Successful transformants can be selected for in known manner, by takingadvantage of the attributes contributed by the marker gene, or by othercharacteristics (such as ability to produce lactic acid, succinic acidand/or fumaric acid, inability to produce ethanol, or ability to grow onspecific substrates) contributed by the inserted genes. Screening can beperformed by PCR or Southern analysis to confirm that the desiredinsertions and deletions have taken place, to confirm copy number and toidentify the point of integration of genes into the host strain'sgenome. Activity of the enzyme encoded by the inserted gene and/or lackof activity of enzyme encoded by the deleted gene can be confirmed usingknown assay methods.

The deletions or inactivations envisaged herein can be accomplished bygenetic engineering methods, forced evolution or mutagenesis and/orselection or screening. Indeed, the present state of the art provides awide variety of techniques that can be used for the inactivation,deletion or replacement of genes. Such molecular techniques include butare not limited to:

(i) gene inactivation techniques based on natural gene silencing methodsincluding antisense RNA, ribozymes and triplex DNA formation,(ii) techniques for single gene mutation such as gene inactivation bysingle crossing over with non-replicative plasmid and gene inactivationwith a non replicative plasmid or a linerized DNA fragment capable ofdouble-crossover chromosomal integration (Finchham, 1989,Microbiological Reviews, 53: 148-170; Archer et al., 2006, BasicBiotechnology: 95-126), and(iii) techniques for multiple unmarked mutations in the same strain,such as but not limited to:(a) deletion and replacement of the target gene by an antibioticresistance gene by a double crossover integration through homologousrecombination of an integrative plasmid, giving segregationally highlystable mutants;(b) removing of the antibiotic resistance gene with the Flp recombinasesystem from Saccharomyces cerevisiae allowing the repeated use of themethod for construction of multiple, unmarked mutations in the samestrain and(c) generating a strain deleted for the upp gene, encoding uracilphosphoribosyl transferase, thus allowing the use of 5-fluorouracyl as acounter selectable marker and a positive selection of the doublecrossover integrants.

In particular embodiments the deletion or disruption of the endogenousgene is performed according to the method described by Oliveira et al(2008) (Appl Microbiol Biotechnol 80, 917-924)

In particular non-limiting embodiments of the present invention, thedeletion or disruption of the endogenous gene, may include theintroduction of one or more functional structural genes, notably a geneencoding an enzyme involved in the production of the organic acid ofinterest, such as an LDH gene as described above, inserted between the5′ and 3′ flanking portions of one of the endogenous genes of the hoststrain. The functional gene preferably includes functional promoter andterminator sequences operatively linked to the structural gene. Thisapproach allows for the simultaneous deletion of the endogenous gene andinsertion of the functional exogenous or heterologous gene. The vectormay include a selection marker gene instead of or in addition to thestructural gene. Again, the selection marker gene is positioned on thevector between the 5′ and 3′ flanking portions of the endogenous gene(s)being targeted, and becomes inserted in the locus of the functionalendogenous gene. The use of a selection marker gene has the advantage ofintroducing a means of selecting for successful transformants. However,it is also possible to select for successful transformants based on theresulting functional characteristics. For instance, depending on thegenes deleted and introduced it may be possible to screen on reduced oreliminated ability to grow on specific building blocks, to produce theorganic acid of interest at high concentrations or on their reducedability to produce specific metabolites such as ethanol.

Accordingly, a further aspect of the present invention provides methodsof obtaining high yield organic acid producing micro-organisms, whichmethods comprise

-   -   a) obtaining a micro-organism of the genus of Monascus having a        high tolerance to the organic acid at low pH;    -   b) transforming the micro-organism with one or more recombinant        nucleic acid sequences which ensure an increased production of        the organic acid and/or a reduction of endogenous production of        metabolites; and    -   c) Selecting a micro-organism capable of high yield organic acid        production.

The step of identifying a micro-organism having a high tolerance to theorganic acid at low pH can be obtained by selecting the micro-organismon a medium containing high concentrations of the organic acid. Moreparticularly selection is performed by selection on a medium containingthe organic acid at a pH which is less than 1.5 unit more than the(lowest) pKa value of the relevant organic acid. In particularembodiments the pH is less than one unit more than the relevant pKavalue. In further particular embodiments the pH is less than the pKavalue.

In particular embodiments, the micro-organisms are selected on a mediumcontaining the organic acid at 50 g/L, most particularly 100 g/L, and inparticular embodiments the micro-organism are selected on a mediumcontaining the organic acid of up to 150 to 175 g/L In particularembodiments, the organic acid is lactic acid, succinic acid and/orfumaric acid, and the pH is less than 3.8, more particularly less than3.

It has surprisingly been found by the present inventors thatmicro-organisms of the order of Monascus can be identified which aretolerant to high organic acid concentrations, such as, but not limitedto high lactic acid, succinic acid and/or fumaric acid concentrations,at a low pH, more particularly at a pH which is less than the pKa of theorganic acid. More particularly it has been found that micro-organismsof the order of Monascus can be identified which are tolerant toincreased lactic acid, succinic acid and/or fumaric acid concentrationsat a pH of less than 3.0.

The step of transforming the micro-organism is described in detailhereinabove. As detailed above, different genetic modifications areenvisaged which increase the yield of organic acid production.

The step of selecting a micro-organism capable of high yield organicacid production is a selection step known to the skilled person andincludes but is not limited to measuring activity of enzymes involved inthe production of the organic acid of interest by methods such as thosedescribed for LDH in the Examples herein. Additionally or alternativelyin the methods according to the invention, the selection step can bebased on reduced production of ethanol, reduced organic acidconsumption, etc. . .

In a further aspect, the present invention provides methods of producingan organic acid, more particularly at high yield. Indeed, high yieldproduction of organic acids is of interest in view of its numerousindustrial applications. The methods of the present invention are ofinterest for the production for organic acids as food or feed additiveas acidulant, flavoring agent, pH buffering agent, or preservative, foruse in pharmaceuticals and cosmetics and for the production of detergentand polymers. Lactic acid polymers for instance have properties similarto petroleum-derived plastic but have the advantage of beingbiodegradable and environmentally friendly.

In particular embodiments, the methods of the present invention comprisethe steps of obtaining a micro-organism of the order of Monascus whichis tolerant to high concentrations of organic acid at low pH,genetically modifying it to increase yield of the organic acid andculturing the thus obtained micro-organism in the presence of specificsubstrates or chemical building blocks at a pH of less than 5, moreparticularly less than 4, more particularly at a pH which is less than1.5 units above the pKa of the organic acid. In particular embodiments,the methods of the present invention comprise the steps of

(i) obtaining a strain of the order of Monascus, which is tolerant tothe organic acid at low pH, more particularly at a pH which is less than1.5 units above the pKa of the organic acid; (ii) modifying said strainsuch that it is capable of producing the organic acid of interest athigh yield from hexose or pentose sugars or combinations of hexose andpentose sugars; and (ii) culturing said strain in the presence of asuitable substrate, more particularly at a pH which is less than 1.5units above the pKa of the organic acid, most particularly at a pH ofless than 5, most particularly less than 4.

In particular embodiments of the methods of the present invention, theproduced organic acid is a carboxylic acid, even more particularlylactic acid, succinic acid and/or fumaric acid. Most particularly, theproduced organic acid is L-lactic acid.

Methods for obtaining a micro-organism tolerant to organic acids of theorder of Monascus and methods of modifying said organism to increaseorganic acid production yield are described hereinabove and illustratedin the Examples section.

In particular embodiments of the process of the invention, themicro-organism or strain of the order of Monascus are cultivated in amedium that includes a sugar that is fermentable by the transformedstrain. The sugar may be a hexose sugar such as glucose, glycan or otherpolymers of glucose, glucose oligomers such as maltose, maltotriose andisomaltotriose, panose, fructose, and fructose oligomers. In particularembodiments, the micro-organism is modified to have the ability toferment pentose sugars, and the medium includes a pentose sugar such asxylose, xylan or other oligomer of xylose. In particular embodiments,the organisms are cultivated on combinations of hexose and pentosesugars.

The sugars can be hydrolysates of a hemicellulose orcellulose-containing biomass. In particular embodiments, themicro-organism is modified to ensure degradation of the biomass tomonomers (e.g. expression of cellulase genes). Accordingly, inparticular embodiments, the substrate comprises a sugar oligomer orpolymer such as cellulose, hemicellulose or pectin.

Additionally or alternatively, enzymes can be added to the cultivationmedium to ensure degradation of the substrate into fermentable monomers.

In particular embodiments of the invention, the medium contains at least(the equivalent of) 5 g/L, at least 10 g/L, at least 20 g/L, at least 30g/L, more particularly at least 40 g/L, and even more particularly atleast 50 g/L glucose. In further particular embodiments, the mediumcomprises at least 100 g/L, more particularly at least 200 g/L.

The medium may optionally contain further nutrients as required by theparticular Monascus strain, including inorganic nitrogen sources such asammonia or ammonium salts, and the like, and minerals and the like.However, in more particular embodiments, the medium is a completemineral medium comprising a pentose or hexose sugar as the only carbonsource. The ability of the strains of the present invention to grow onthis simple medium greatly reduces cost of cultivation and simplifiespurification of the organic acid produced. Other growth conditions, suchas temperature, cell density, and the like are not considered to becritical to the invention and are generally selected to provide aneconomical process. Temperatures during each of the growth phase and theproduction phase may range from above the freezing temperature of themedium to about 50° C. A preferred temperature, particularly during theproduction phase, is from about 30-45° C. The culturing step of themethods of the invention may be conducted aerobically, microaerobicallyor anaerobically. Quasi-anaerobic conditions or oxygen limitedconditions, in which no oxygen is added during the process but dissolvedoxygen is present in the medium at the start of the production process,can also be used.

In particular embodiments, the methods of the present invention comprisecultivation of micro-organisms (strains) of the order of Monascus whichexhibit the ability to convert sugars to an organic acid under anaerobicor oxygen-limited conditions.

The cultivation step of the methods according to this aspect of theinvention can be conducted continuously, batch-wise, or some combinationthereof.

The yield of organic acid obtained by the tools and methods according tothe present invention will depend on the cultivation conditions used.

In certain embodiments, the methods of producing an organic acidaccording to the present invention result in a yield of organic acidthat is at least 0.5 g/L, particularly at least 2 g/L, more particularlyat least 4 g/L, even more particularly at least 50 g/L, and mostparticularly between 50-100 g/L. In further particular embodiments theproduction yield of about 2-3 g/L/hour.

In particular embodiments the micro-organisms of the present inventionare capable of converting at least 50%, more particularly at least 60%,even more particularly 75%, most particularly at least 95%, and up to100% of the glucose consumed. In practice, the yield obtained inparticular embodiments of the methods of the present invention is atleast 0.5 g/g sugar, more particularly at least 0.6 g/g sugar, but maybe up to 0.95 g/g sugar.

In further embodiments, the invention provides methods for producing anorganic acid which, in addition to the steps detailed above furthercomprise the step of recovering the organic acid of interest. Inparticular embodiments, recovery of the organic acid from cultivationmedium in the methods of the present invention is greatly simplified inview of the fact that the organisms can be grown on a mineral mediumcontaining only sugars as a carbon source. Suitable purification can becarried out by methods known to the person skilled in the art such as byusing extraction, ion exchange resins, electrodialysis, nanofiltration,etc. . .

The present invention will now be further illustrated by means of thefollowing non-limiting examples.

EXAMPLES Example 1 Isolation of Three Monascus ruber Strains LF4, LF5and LF6 as Highly Organic Acid Tolerant Strains

The strains were isolated from soil and corn-and-wheat silage byincubation in medium at low pH, increasing lactic acid concentration andvarying concentrations of glucose and xylose concentrations.

One strain (LF4) was isolated by incubation in DSMZ402 medium with 50g/l glucose, 50 g/l xylose and 100 g/l lactic acid at pH 2.8 at 40 C.

Another strain (LF5), was isolated as described for LF4, but at pH 2.4

A third strain (LF6), was isolated by incubation in DSMZ402 medium with25 g/l xylose and 150 g/l lactic acid at pH 2.8 and 32 C.

These three strains of the order of Monascus, more particularly Monascusruber were found to be able to grow at low pH (2-3) in the presence ofhigh lactic acid concentrations (up to 150 g/L).

Lactic acid tolerant strains LF4, LF5 and LF6 were deposited as depositunder the Budapest Treaty by the Food & Biobased Research department ofthe Stichting Dienst Landbouwkundig Onderzoek, Bomseweilanden 9, 6708 WGWageningen, Nederland on Jul. 21, 2010 and were attributed depositnumbers CBS 127564, CBS 127565 and CBS 127566, respectively.

Example 2 Stable Transformation with a Selectable Marker Gene Materialsand Methods a) Transformation

Most protocols for genetic modification of Monascus ruber involve thetransformation of protoplasts using electroporation or by combination ofCaCl₂ and polyethylene glycol. However these methods often had lowtransformation efficiency and low mitotic stability. A transformationmethod using Agrobacterium tumefaciens-mediated integration of DNA withhigh efficiency has been reported in M. ruber (Yang 2008). We have usedthis Agrobacterium tumefaciens-mediated transformation system for our M.ruber strains LF4, LF5 and LF6 and two selected CBS strains CBS 135.60and CBS 503.70. Agrobacterium strains containing the binary vector pUR5750, described by de Groot et. al, 1998, were obtained from PlantResearch International, WUR. These were grown at 30° C. for 48 h inminimal medium supplemented with kanamycin (100 μg/ml). The cells(OD₆₀₀˜1.0) were washed with induction medium without Acetosyringone(AS) and grown with and without AS for 6 h at 28° C. AS was used forinduction of virulence and T-DNA transfer. Conidiaspores of Monascusruber (10⁷ spores/ml) were collected from PDA plates after 10 days ofculture. When conidia were transformed, an equal volume of conidia wasmixed with an equal volume of A. tumefaciens and plated out on nylonfilters placed on induction medium with and without AS. The plates wereincubated at 25° C. for 4 days. The filters were then transferred toYM-medium containing 200 μg/ml cefotaxime to kill the Agrobacteriumcells and 100 μg/ml hygromycin to select for transformants.

After 10-14 days fast growing fungal colonies were transferred to freshYM plates+hygromycin and cefotaxime. After 5 days of growth the edge ofthe colony was transferred again to a fresh YM plates+hygromycin andcefotaxime.

Applying this procedure results in hygromycin resistant transformantsfor each of the selected Monascus strains (see Table 2).

TABLE 2 Number of transformants per M. ruber strain M. ruber strain No.of transformants LF4 14 LF5 3 LF6 2 CBS 503.70 4

b) Stability of the Transformants

The first property of the transformants we have addressed is the geneticstability. With respect to this, two features of genetically modifiedstrains are important:

(i) the target gene has to be integrated into the genome, and(ii) the gene has to stay in place and active even without the selectionpressure of the antibiotic

Presence of the vector DNA in the transformants was first shown by meansof PCR.

DNA was isolated from 7 transformants (5 LF4 transformants and 2 CBS503.70 transformants) and from three wild type strains (LF4, CBS 503.70and CBS 135.60) and subjected to a PCR with DNA primers able to show thehygromycin gene.

The DNA of the transformants clearly yield a PCR fragment of the samesize as from the control vector DNA while the wild type strain do notshow this PCR fragment. This shows the presence of the hygromycin genein the transformants.

In order to establish integration of the vector DNA in the genomic DNAfrom the M. ruber transformants a Southern blot analysis was performed.Genomic DNA was blotted before and after digestion with a restrictionenzyme. The blot was hybridized with a probe showing the presence of thehygromycin gene.

In the lanes with the genomic DNA the signals on the blot coincide withthe position of the genomic DNA meaning integration of the hygromycingene in the genome. In the lanes with the digested DNA the signals arevisible on different positions (=different DNA fragments) meaning randomintegration of the hygromycin gene.

In conclusion, we have established random genomic integration of thevector DNA in the M. ruber strains. In order to test the stability ofthe transformants without the selective pressure of the antibiotic thestrains were grown on PDA (potato dextrose agar) plates. After growthfor approx. 14 days a part of the outer edge of the culture wastransferred to a fresh PDA plate again without hygromycin. This processwas repeated 3 times. Finally a part of the outer edge of the culturewas transferred to a fresh PDA plate with hygromycin to see whether thestrain was still able to grow in the presence of this antibiotic.

The number of transformants still able to grow on selective plates afterthis procedure was scored (Table 3).

TABLE 3 M. ruber transformants screened for the stability of thehygromycin resistance. M. ruber No. of transformants No. oftransformants growing strain tested on Hyg. after 3 transfers LF4 14 14LF5 3 3 CBS 503.70 4 4

A second approach was to collect spores from the plate after 3 transferswithout antibiotic selection and compare the number of coloniesappearing after spreading the spores on PDA plates with and withouthygromycin (selective plates). The spores were filtered through glasswool to minimize contamination with mycelial fragments. If thehygromycin gene is lost or inactivated the number of colonies onselective plates is reduced.

A test with all strains showed no reduction in the number of colonies onselective and non-selective plates. In conclusion, there is no evidencefor instability or inactivation of the introduced gene in the M. rubertransformants.

As a final test the Southern blot experiment described before has beenrepeated with 27 transgenic strains after the sequential transfer onnon-selective plates and subsequently growth in medium without selectivepressure.

This Southern blot confirmed the presence of the hygromycin gene in thegenome of the M. ruber transformants after growth in non selectivemedium.

c) Construction of Transformation Vectors

In order to be able to perform sequential transformations on M. ruberstrains for instance to introduce multiple genes and/or to combineintroduction of genes with knock out events, we set on to constructtransformation vectors with other selectable markers.

The vector that has been used in the transformation described in theprevious paragraph is the pUR5750 plasmid. FIG. 1 shows a map of thisplasmid (“RB”=right border; “LB”=left border; “Hpt”=hygromycinphosphotransferase gene).

During transformation the DNA part between the RB (right border) and theLB (left border) is transferred into the fungal genome. On this part theHpt gene is located. The activity of this gene, under regulation of thegpd-promoter and trpC-terminator, results in hygromycin resistance intransformants. In order to facilitate cloning and exchange of promoters,genes and terminators in this vector we designed a cassette model. Asshown in FIG. 2, this cloning strategy enables us to exchange promoters,terminators and genes in the vector but also to combine 2 gene cassettesin a row.

Hpt is the hygromycin phosphotransferase gene giving hygromycinresistance which we already have used. Ble is the bleomycin gene givingphleomycin or zeocin resistance. NptII is the neomycinphosphotransferase gene giving neomycin (G418) resistance.

Since the pUR5750 transformation vector is a large vector, which willbecome even larger when additional genes are inserted (see FIG. 2), wedecided to use also a shorter version of this vector for thetransformation of M. ruber. In general it is assumed that smallervectors can be handled more easily during construction andtransformation experiments.

This smaller vector is a derivative of the pUR5750 and is coded pCB301(Xiang et al. 1999). Typical DNA elements necessary for planttransformation, the original function of the basic sequence of pUR5750,have been removed from this plasmid leaving a 3574 bp vector. FIG. 3shows the pCB301 containing the Hpt cassette called pCGHT3 (“RB”=rightborder; “LB”=left border; “Hpt”=hygromycin phosphotransferase gene).This vector is half the size of the original Hpt vector.

In order to be able to use a selectable marker like the Ble or the NptIIgene we first confirmed inability of Monascus ruber to grow in mediacontaining concentrations of Zeocin or G418 mostly used in fungaltransformation systems. Conidia and spores from Monascus ruber strainLF4 and LF6 were not able to grow on PDA plates containing 600 μg/mlZeocin or 200 μg/ml G418.

Using the two basic vectors and three selectable markers six vectorswere made (Table 4).

TABLE 4 The vector constructs made Basic Marker Basic Marker vector geneConstruct vector gene Construct pUR5750 Hpt pURGHT2 pCB301 Hpt pCGHT3Ble pURGBT1 Ble pCGBT2 NptII pURNT3 NptII pCGNT13d) Transformation with the Six Newly Constructed Vectors

A mixture of conidia and ascospores from M. ruber strain LF5 wastransformed with the six vectors as described before. After thecocultivation step the filters with the outgrowing conidia andascospores were transferred to selective plates.

The selective plates contain YM-medium with 200 μM cefotaxime to killthe Agrobacterium cells and either 100 μg/ml Hygromycin, or 200 μg/mlZeocin, or 200 μg/ml Geneticin to select for transformants.

After approx. 12 days the first growing colonies became visible on theselective plates.

Table 5 shows the score of the transformants.

TABLE 5 Transformants obtained from LF5 conidia. Vector Marker gene No.of transformants pURGHT2 Hpt 7 pURGBT1 Ble  0* pURGNT3 NptII 1 pCGHT3Hpt 18  pCGBT2 Ble  0* pCGNT3 NptII 9 *General disperse growth, nospecific colonies visible.

Colonies were transferred to PDA plates with appropriate antibiotics andafter sufficient growth used as an inoculum for growth in liquid medium(YPD) containing the antibiotic. Mycelium was harvested after 4 days andDNA was isolated.

In order to establish the integration of the selectable marker gene inthe genome from the M. ruber LF5 transformants two PCR analysis wereperformed on a number of transformants. The first analysis which willshow the presence of the selectable marker in the isolated DNA is a PCRwith primers specific for the marker genes. The second PCR analysiswhich excluded the presence of contaminating vector DNA, possibly comingfrom surviving A. tumefaciens bacteria, was performed with primersspecific for the A. tumefaciens GPD (glyceraldehyde phosphatedehydrogenase) gene.

In conclusion it has been shown that Hpt and NptII vectors which havebeen constructed according to the cassette strategy are effective intransforming M. ruber. Using the smaller pCB301 based vector seems toresult in more transformants than the pUR5750 based ones. PCR analysisshows integration of the selectable marker genes in the genome of M.ruber and also shows that A. tumefaciens bacteria have efficiently beenkilled by the Cefotaxim.

Example 3 Generation of a Lactic Acid Producing Monascus a) VectorConstruction

For the introduction of at least one copy of the Bovine (Bos taurus) LDHgene in the genome of the Monascus ruber strains, the moststraightforward approach is introducing a codon optimized Bovine LDHgene in the cassette as described hereabove. The promoter and terminatorsequences driving the expression of the LDH gene will be the same hasthose driving the selectable marker gene.

For codon optimization we first analyzed the codon usage of Monascus.Since only two genes from M. ruber are available we compared the codonusage of M. pilosus and M. purpureus with the codon usage of the M.ruber genes. The three species are closely related and a great deal ofsimilarity between the codon preference of the three Monascus specieswas found.

Based on the codon usage we were able to design a optimized Bovine LDHgene for expression in the M. ruber strains selected (SEQ ID NO:1 andFIG. 4) and had such a gene synthesized by Genscript Corporation. Afterobtaining this gene it was cloned into both type of Hpt vectorsconstructed and tested. The cloning was performed according to thecassette strategy shown in FIG. 2 which has the advantage that theoriginal vector is unchanged except for the insertion of thepromoter-LDH-terminator cassette. This results in vectors pCGHTGBtLTbased on the pCB301 plasmid (FIG. 5) and pURGHTGBtLT based on thepUR5750 plasmid (FIG. 6).

b) Cloning and Expression of the Bt-LDH Gene in E. Coli.

In order to confirm the correct translation and the activity of thesynthetic LDH gene used for transforming M. ruber, the gene was clonedin an E. coli expression vector. The expression vector used is theInVitrogen pBAD102 vector which will express the protein after Arabinoseinduction.

LDH was highly expressed in E. coli but was found to be partly soluble.

The soluble E. coli proteins were used to analyse the LDH enzymeactivity.

The reaction catalyzed by the btLDH is:

(L)-lactate+NAD(+)<=>pyruvate+NADH

In the activity assay an excess of pyruvate and NADH is added so theactivity is reflected by the conversion of NADH to NAD which canspectrofotometrically be measured at 340 nM.

As a positive control LDH enzyme obtained from Sigma-Aldrich was used.

The reaction velocity was determined by a decrease in absorbance at 340nm resulting from the oxidation of NADH. One unit causes the oxidationof one micromole of NADH per minute at 25° C. and pH 7.3, under thespecified conditions.

0.2 M Tris.HCl buffer was prepared. The LDH enzyme was diluted prior touse to obtain a rate of 0.02-0.04 ΔA/min. in Tris buffer and kept cold.

A reaction mix of 2.8 mL Tris.HCl, 0.2 M pH 7.3 and 0.1 mL 6.6 mM NADHwas prepared.

LDH activity was measured with and without substrate added, in order tomonitor background NADH conversion.

The analysis of the expression and activity of the synthetic LDH in E.coli shows that the synthetic gene encodes a protein of the correct Mwand with LDH activity. The glucose and lactose production by these E.coli strains over time are shown in FIG. 7 (“Coli-LDH 1, 2, and 3”correspond to three independent E. coli extracts with the expressedsynthetic LDH protein present; “Coli-control” is an E. coli extractexpressing a different protein).

c) Transformation of Monascus ruber with the LDH Vectors.

A mixture of conidia and ascospores from M. ruber strains LF4, LF5 orLF6 was used for transformation with the LDH vectors pCGHTGBtLT andpURGHTGBtLT. So in total 6 transformations were performed. Afterapplying the transformation procedure as described before and selectionon hygromycin containing plates only three LF5 transformants wereobtained.

One transformant resulted from the use of the pCGHTGBtLT vector (LF5-t1)and two from the pURGHTGBtLT vector (LF512, LF5-t5).

d) Analysis of M. Ruber LF5-T1 and LF5-T2-LDH Transformants

In a first experiment, two transformants LF5-T1 and LF5-T2 wereanalysed.

LF5-T1 was transformed with pCGHTGBtLT, and LF5-T2 with pURGHTGBtLT.

In order to analyze the LDH enzyme activity and possible L-lactateproduction the two transformants and the wt-LF5 strain were grown inS.c. medium+50 g/L glucose pH 6.0.

Medium and biomass samples were taken from individual cultures after 24,48, and 72 hours of cultivation.

(i) Glucose Consumption and Lactate Production

FIGS. 8 a and 8 b clearly show glucose consumption in all cultures whilein the medium from LF5-T2 lactic acid is detected increasing inconcentration with time (the numbers 24, 48 and 72 indicate samples fromtwo separate cultures grown for 24, 48 and 72 hours; “M” corresponds tomedium). LF5-T2 consumed approximately 8 g/L glucose and producedapproximately 1.5 to 2 g/L of lactic acid, indicating a yield between0.18 and 0.25 g/g was reached. The maximum theoretical yield is 1.00g/g.

(ii) Enzymatic Analysis

Since the HPLC analysis does not discriminate between L- and D-lacticacid a number of samples was analyzed by an enzymatic method. A L-lacticacid analysis kit (Megazyme) confirmed the presence of L-lactic acid inthe LF5-T2 samples (FIG. 9).

The harvested biomass samples were frozen in liquid nitrogen and groundusing a mortar and a pestle. The frozen powder was thawed in buffer (0.2M Tris HCl, pH 7.3) and the protein was extracted by vortexing. Aftercentrifugation the supernatant was subjected to protein analysis and toan LDH-enzyme activity analysis as described in the previous paragraph.

This analysis clearly showed the presence of L-LDH activity in theLF5-T2 transformant, whereas no activity was found in the control (FIG.9).

(iii) Southern Blot Analysis

Hybridization was performed using the LDH gene. Southern blot analysisconfirms a single integration of the LDH gene in the genome of the M.ruber transformant t2, after growth in non selective medium.

Transformant t1 was found not to contain the LDH gene. A second Southernanalysis with the hygromycin gene shows integration of the hygromycingene in both transformants. This indicates that in transformant t1, thegene cassettes, LDH and hygromycin between both borders was notcompletely integrated.

(iv) Conclusion

Using the transformation vectors constructed with a combination of aselectable marker and a synthetic LDH gene 27 M. ruber transformantswere obtained. One transformant, LF5-T2 shows production of lactic acidwhen grown on glucose medium.

Lactic acid production, L-LDH activity and southern blots support theconclusion that we have inserted an L-LDH encoding heterologous gene inthe genome of this M. ruber LF5-T2 transformant in such a way that it isexpressed and metabolically active.

e) Analysis of M. Ruber LF4-, LF5-, and LF6-LDH Transformants

In a next experiment, a total of 35 transformants were analyzed, i.e. 4of LF4, 16 of LF5 and 15 of LF6. Strains were precultured in 10 ml YEPDmedium for 3 days. Biomass was washed in Sc medium with 10 g/L glucoseat pH 2.8 and was cultivated in 15 ml of Sc medium with samecomposition. The glucose consumption by the strains in illustrated inFIG. 10. Nineteen (54%) of these transformants, i.e. two derived fromLF4, twelve derived from LF5 and five derived from LF6, produced lacticacid from glucose (FIG. 11). In the positive experiments, varyingamounts of lactic acid were found, ranging from 0.5 to 3.3 g/L,corresponding to 5-33% of the maximal theoretical yield (Figure).Ethanol was formed as byproduct, its concentration inversely related tothe lactic acid concentration (FIG. 12).

Two transformants of each strain were selected (4t3, 4t4, 5t2, 5t21,6t4, 6t5), the one with the highest lactic acid production and one withthe lowest ethanol production. The strains were subsequently cultivatedin shake flasks (on either glucose or xylose (both 10 g/L)), under twodifferent aeration conditions: aerobic and severely oxygen limited.

Under aerobic conditions, strains were precultured in 100 ml shakeflasks on 10 ml YEPD for three days. The cultures were then washed twicewith 25 ml Sc medium, at pH 2.8. Biomass was homogenized in 10 ml of thesame medium using a triangular magnetic stirring rod. 750 microliterswere used to inoculate 15 ml of Sc medium, pH 2.8, containing 10 g/L ofglucose or xylose (FIGS. 13-15).

Alternatively, under severely oxygen limited conditions, cultures wereperformed in bottles closed with an aluminium capped butyl rubberstopper equipped with bicycle tube valves to create severely oxygenlimited, almost anaerobic conditions. Precultured in 100 ml shake flaskson 10 ml YEPD during 3 days. The cultures were then washed twice with 25ml S.c medium, at pH 2.8. Biomass was homogenized in 10 ml of samemedium using a triangular magnetic stirring rod. 750 microliters wereused to inoculate 15 ml of Sc medium, pH 2.8, containing 10 g/L ofglucose or xylose.

In the aerobic cultures (left panels of FIGS. 13-15)) some lactic acidwas produced (approximately 0.5-1 g/L) from glucose. The lactic acid wasconsumed after the glucose was fully consumed. No ethanol was formed.Under severely oxygen limited conditions (Right panels of FIGS. 13-15))the lactic acid concentration was between 2.0 and 3.3 g/L, and ethanolwas produced at concentrations varying between 0.5 and 1.0 g/L.

Example 4 Further Improvement of Lactic Acid Yield by EliminatingEthanol Production a) Identification of the Pyruvate Decarboxylase GenesUsing Sequence Homology

In order to further improve lactic acid yield a gene knock-out systemwas envisaged to reduce the production of products other than lacticacid on the one hand and to reduce the metabolism of lactic acid byMonascus on the other hand. More particularly, in order to avoid theproduction of alcohol, a knock-out system was envisaged based on genesencoding M. ruber homologues of the S. cerevisiae PDC1 gene encodingpyruvate decarboxylase (EC 4.1.1.1).

M. ruber produces ethanol under oxygen-limited conditions. Since bothethanol and lactic acid are produced from pyruvate, the production ofethanol should be prevented since it decreases the yield of lactic acid.

Using BLAST with the S. cerevisiae PDC1 gene as the query we foundseveral homologues in the Aspergillus niger genome which could representa PDC encoding gene. A. niger was used because of its close relationshipto Monascus. Based on these sequences we designed degenerated PCRprimers in order to be able to clone part of a M. ruber PDC.

RT-PCR on RNA from M. ruber strain LF6 resulted in two 1.3 kB DNAfragments which were cloned in a plasmid. Sequencing of these cloned PCRfragments confirmed the presence of two open reading frames with highhomology to the Aspergillus niger PDC homologues and sufficient homologyto the S. cerevisiae PDC1 gene to conclude that we have cloned two PDC1analogues of Monascus ruber.

The open reading frame for the M. ruber PDC genes PDC1 and PDC2 isprovided as SEQ ID NO:3 and 4 (and FIGS. 16 and 17).

b) Construction of a Gene Knock-Out Vector. i) Knock-Out of PDC Genes

Using PCR, the 5′ and 3′ halves of the PDC1 and PDC2 genes were isolatedand suitable restriction sites are added to the ends. The 5′ halves arethen cloned 5′ of the promoter-marker-terminator cassette of vectorspCGNT1 and pCGBT2 (and 3′ of the Right Border sequence). These vectorsare identical to the previously described vector pCGHT3, except that theNPTII and BLE genes are used as selectable markers instead of the HPTgene. Likewise, the corresponding 3′ halves are cloned 3′ of thepromoter-marker-terminator cassette (and 5′ of the Right Bordersequence). In this manner, three vectors are generated:

1. pPDC1::NPT for disruption of PDC1 using geneticin selection (FIG. 18)2. pPDC2::NPT for disruption of PDC2 using geneticin selection (FIG. 19)3. pPDC2::BLE for disruption of PDC2 using zeocin selection (FIG. 20)

These vectors are used in a previously constructed PDC1::NPT disruptant

ii) Combined Knock Out and Gene Insertion

Vectors were constructed to enable simultaneous disruption of a targetgene and introduction of another gene. In order to minimize unwantedrecombination in a second round of transformation due to the presence oflong stretches of DNA in the vector and in order to increase the cloningefficiency during vector construction the size of the promoter andterminator sequences were reduced in the new vectors. Using PCR the sizeof the GPD promoter was reduced from 2208 bp to 538 bp and the TrpCterminator was reduced from 763 bp to 278 bp. These are vector pCH#PDC1and vector pCL1H#PDC1 (see FIGS. 21 and 22). The pCH#PDC1 vector is usedfor the disruption of PDC1 using hygromycin selection. The pCL1H#PDC1vector is used for the disruption of PDC1 and introduction of LDH usinghygromycin selection. Transformants resulting from these vectors have adisrupted PDC1 gene and a disrupted PDC1 gene+an inserted LDH generespectively. The vector pCN#PDC2 was constructed to disrupt the PDC2gene using geneticin selection (FIG. 23).

c) Generating Knock Out Transformants i) Knock-Out of PDC Genes

Spores obtained from LF6 LDH transformant t4 and t5 are used intransformation experiments with the knock out vectors pPDC1::NPT orpPDC2::NPT. After selection on G418 (geneticin) each combination ofstrain and vector results in 50-80 geneticin resistant colonies. Bymeans of PCR with gene specific primers the DNA of 40 transformants (20LF6t4+pPDC2::NPT transformants and 20 LF6t5+pPDC2::NPT transformants)are tested for the presence of the NPTII gene and for the disruption ofthe PDC2 gene.

Disruption of the PDC2 gene with the knock out vector is observed.

ii) Combined Knock Out and Gene Insertion

The vectors pCH#PDC1 and pCL1H#PDC1 were used to transform the spores,collected from the wild type strain LF6 at 40° C., according to ourstandard procedure. After 10 days of growth on selective platescontaining hygromycin growing colonies were observed and 20 of them weretransferred to new hygromycin containing plates and tested by PCR forPDC1 gene knock out.

PCR analysis shows that in 1 out the 20 transformants from pCH#PDC1 andin 3 out of 20 transformants from pCL1#PDC1 the PDC1 gene has beendisrupted (Table 6).

TABLE 6 Number of transformant per construct construct Colonies totalColonies transferred PDC1 knockout pCH#PDC1 53 20 1 pCL1H#PDC1 97 20 3

The vector pCN#PDC2 was used to transform spores from a PDC1 knock outstrain from transformation round 1. After 10 days of growth on selectiveplates containing geneticin growing several colonies were observed and40 of them were transferred to new geneticin containing plates andtested by PCR for PDC2 gene knock out.

Strain LF6KL19 was confirmed to be a double knockout of PDC1 and PDC2and contains the recombinant LDH gene.

d) Analysis of Transformants

Strain LF6KL19 was precultivated in 300 ml Sc medium containing 50 g/lglucose and 1 g/l Junlon in a 2 L erlenmeyer at pH 2.8. The initialspore concentration was 1×10⁵ spores/ml. The incubation temperature was30 or 35° C. and agitation speed was set at 75 rpm. At 30° C. 16.1 g/lglucose was consumed and 14.4 g/l lactic acid was produced, at 35 18.8g/l of glucose was consumed and 18.6 g/l lactic acid was produced,indicating the actual yield was between 0.9 and 1.0 g/g. Under theseconditions also the highest productivities were obtained: 0.15 g/l/h.

e) Genome and Transcriptome Analysis

The genome of M. ruber LF6 and the transcriptomes of M. ruber LF6 and M.ruber LF6KL19 (double PDC knock-out, introduced copy of bovine LDH),growing on different growth substrates, were sequenced and the relevantgenes involved in alcohol production were identified on the genome by anautomated annotation procedure. The RNA sequence data were used toimprove the architecture of these genes. Many putative genes that areinvolved in the formation of ethanol were identified on the genome: 7for pyruvate decarboxylases (PDCs) and 12 for alcohol dehydrogenases. 3putative PDC genes were adjacent to each other in the genome, Analysisof the NA sequence data showed that these 3 putative PDC genes belong toone single gene. The PDC2 product (SEQ ID NO:2) was identical to part ofthe translated sequence of this gene.

The PDC1 product was identical to part of the putative PDC gene productof Mona10180. Both genes encoding PDC1 and PDC2 were highly expressedunder all circumstances. Besides these, also another PDC gene (Mona07809or PDC4, SEQ ID NO: 5, FIG. 24) was transcribed under all circumstancesand can be eliminated from the genome of M. ruber to reduce theproduction of ethanol.

Example 4 Further Improvement of Lactic Acid Yield by Eliminating ofEndogenous Lactic Acid Metabolism a) Cyt-LDH Activity Analysis in M.Ruber

From literature it is known that in fungi and yeasts lactic acid ismetabolized by a (cytochrome)L-lactate dehydrogenase. Therefore we firstanalyzed M. ruber strains grown on lactic acid for this enzyme activity.M. ruber strains were grown on 2% yeast extract, 1% peptone mediumsupplemented with 5% glucose or with 2% lactic acid as carbon source. Asa control S. cerevisiae was grown under the same conditions (Lodi andGuiard, 1991). Biomass was harvested after 24 h of growth. The harvestedbiomass samples were frozen in liquid nitrogen and ground using a mortarand a pestle. The frozen powder was thawed in buffer (0.067 M sodiumphosphate, pH 7.4 with 0.001 M EDTA) and the protein extracted byvortexing. After centrifugation the supernatant was subjected to proteinanalysis and to cyt-LDH-enzyme activity analysis. The rate of reductionof potassium ferricyanide is determined spectrophotometrically at 420nm. Prior to assay, the enzyme was dissolved in 0.067 M phosphatebuffer, pH 7.4 with 0.001 M EDTA to obtain a rate of 0.02-0.04ΔA/minute.

A mix of 2.0 ml of 0.1 M sodium pyrophosphate, 0.5 ml of 0.5 M DL sodiumlactate, 0.3 ml of 0.01 M EDTA, and 0.1 ml of potassium ferricyanide wasprepared and pipetted into a micro-titer plate. 10-20 μl ofappropriately diluted sample was added and the ΔA420/min was recorded.

FIG. 25 shows the cyt-LDH activity in the protein samples. “Gluc” meansgrown in medium with glucose and “Lact” means grown in medium withL-lactic acid as a carbon source. Cyt-LDH activity is present in alltested M. ruber strains and is induced by growth on L-lactic acid as acarbon source.

b) Isolation of M. Ruber Homologues of the CYB2 Gene Based on SequenceHomology

Using BLAST with the S. cerevisiae CYB2 gene as the query we foundseveral homologues in the Aspergillus niger genome which could representa cyt-LDH encoding gene. A. niger was used because of its closerelationship to Monascus. Based on these sequences we designeddegenerated PCR primers in order to be able to clone part of a M. rubercyt-LDH.

PCR on genomic DNA from M. ruber strain LF6 resulted in a 1 kB DNAfragment which was cloned in a plasmid. Sequencing of this cloned PCRfragment confirmed the presence of an open reading frame with highhomology to the Aspergillus niger CYB2 homologues and sufficienthomology to the S. cerevisiae CYB2 gene to conclude that we have cloneda CYB2 analogue of Monascus ruber. The open reading frame for M. ruberCYB2 gene is provided as SEQ ID NO:2 and FIG. 26; a putative intron inthe genomic sequence is indicated by small letters.

c) Genome and Transcriptome Analysis of Lactic Acid Metabolism

The genome of M. ruber LF6 and the transcriptomes of M. ruber LF6 and M.ruber KL19 (double PDC knock-out, introduced copy of bovine LDH),growing on different growth substrates, were sequenced and the relevantgenes involved in lactic acid metabolism were identified on the genomeby an automated annotation procedure. The RNA sequence data were used toimprove the architecture of these genes. Several genes encoding lacticacid dehydrogenases were predicted for both isomers of lactic acid. ForL-lactic acid, 4 cytochrome dependent LDH's were predicted, i.e. Mona02475 (partly corresponding to SEQ ID NO:2), Mona 00569, Mona05565 enMona06119. As Mona00569 was found to be the most active and was highlyup regulated in the presence of lactic acid, this is the most obviouscandidate for elimination. The sequence of this gene is given in SEQ IDNo: 6 (FIG. 27).

M. ruber was also found to contain several putative genes for cytochromedependent LDH's that use D-lactic acid as a substrate. Again, one gene(Mona05697) was the most active gene. Since expression of Mona05697 willonly lead to consumption of the undesired D-isomer of lactic acid, it isprobably not necessary to eliminate this gene.

Example 5 Generating a Fumaric Acid Producing Monascus Strain a)Identification of Endogenous Monascus Genes Involved in Fumaric AcidProduction

The genome of M. ruber LF6 was analysed for the presence of sequencesencoding enzymes involved in the production of fumarate. M. ruber wasfound to contain a gene encoding pyruvate carboxylase (SEQ ID NO:7, FIG.28) and a gene encoding malate dehydrogenase (SEQ ID NO:8, FIG. 29).These sequences were selected for the production of fumaric acid in anorganic acid tolerant Monascus strain.

b) Vector Construction.

Vectors are constructed using the methodology described above. Thefollowing vectors are generated:

-   -   pCPCH#PDC1 (FIG. 30), Hygromycin selection, PDC1 knock out,        introduction of Monascus LF6 pyruvate carboxylase gene.    -   pCMDHN#PDC2 (FIG. 31), Geneticin selection, PDC2 knock out,        introduction of Monascus LF6 malate dehydrogenase gene (SEQ ID        NO: 8)    -   pCNATFUM#PDC4 (FIG. 32), Noursethricin selection, PDC4 knock        out, introduction of Rhizopus oryzae fumarase gene codon        optimized for M. ruber (SEQ ID NO: 9, FIG. 33)    -   pCBDAC (FIG. 34), Zeocin selection, introduction of Dicarboxylic        acid carrier gene Mae-1 from Schizosaccharomyces pombe codon        optimized for M. ruber (SEQ ID NO: 10, FIG. 35).

c) Combined PDC Knock-Out and Fumarate Pathway Insertion.

The vectors pCPCH#PDC1, pCMDHN#PDC2, pCNATFUM#PDC4, and pCBDAC are usedto sequentially transform spores collected from Monascus strains at 40°C., according to the standard procedures described above.

First spores from the wild type strain LF6 are transformed with vectorpCPCH#PDC1.

After 10 days of growth on selective plates containing hygromycingrowing colonies are observed and 20 of them are transferred to newhygromycin containing plates and tested by PCR for PDC1 gene knock out(ko) and pyruvate carboxylase gene insertion. This results in LF6-(PDC1ko+PC).

Spores obtained from a LF6-(PDC1 ko+PC) transformant are used fortransformation with the pCMDHN#PDC2 vector. After selection on selectiveplates containing G418 (geneticin) growing colonies are observed and 20of them are transferred to new geneticin containing plates and tested byPCR for PDC2 gene knock out and malate dehydrogenase gene insertion.This results in LF6-(PDC1/2 ko+PC+MD).

Spores obtained from a LF6-(PDC1/2 ko+PC+MD) transformant are used totransformation with the pCNATFUM#PDC4 vector. After selection onselective plates containing nourseothricin growing colonies are observedand 20 of them are transferred to new nourseothricin containing platesand tested by PCR for PDC4 gene knock out and fumarase gene insertion.This results in LF6-(PDC1/2/4 ko+PC+MD+FU).

Spores obtained from a LF6-(PDC1/2/4 ko+PC+MD+FU) transformant are usedto transformation with the pCBDAC vector. After selection on selectiveplates containing zeocin growing colonies are observed and 20 of themare transferred to new zeocin containing plates and tested by PCR fordicarboxylic acid carrier gene insertion. This results in LF6-(PDC1/2/4ko+PC+MD+FU+DAC)=LF6-1-PMFD#PDC.

This sequence of transformations results in strain LF6-1-PMFD#PDCconfirmed to be a PDC1, 2 and 4 knock out and containing a homologouspyruvate carboxylase gene, a homologous malate dehydrogenase, aheterologous fumarase gene and a heterologous dicarboxylic acid carriergene all under control of a constitutive promoter terminatorcombination.

1. A method of producing a composition comprising an organic acidcomprising the steps of: (i) providing a micro-organism of the genusMonascus, tolerant to an organic acid concentration of at least 50 g/Lat a pH of less than 5.0, which has been genetically modified forincreased production of said organic acid; and (ii) culturing saidmicro-organism at a pH which is less than 1.5 unit above the pKa valueof the organic acid in the presence of a hexose or pentose sugars orcombinations thereof as the sole carbon source.
 2. The method of claim1, wherein said micro-organism is tolerant to an organic acidconcentration of at least 50 g/L at a pH of less than 3.0.
 3. The methodaccording to claim 1, wherein said micro-organism comprises one or moreof the following: a) one or more recombinant genes involved in theproduction of said organic acid; and/or b) one or more engineered genedeletions and/or inactivation of genes involved in an endogenousmetabolic pathway which produces a metabolite other than the organicacid of interest and/or wherein the endogenous metabolic pathwayconsumes the organic acid of interest.
 4. The method according to claim1, wherein said micro-organism comprises one or more engineered genedeletions and/or inactivation of genes involved in the endogenousproduction of ethanol.
 5. The method according to claim 4, wherein saidmicro-organism comprises one or more engineered gene deletions and/orinactivation of the endogenous PDC1, PDC2 and/or PDC4 genes. 6.(canceled)
 7. The method according to claim 1, wherein said organic acidis lactic acid and wherein said Monascus strain comprises one or more ofthe following a) a recombinant gene encoding L-LDH; and b) at least oneengineered gene deletion and/or inactivation of an endogenous D-lacticacid production or L-lactic acid consumption pathway or a gene encodinga product involved in an endogenous pathway which produces a metaboliteother than lactic acid.
 8. The method according to claim 7, wherein saidrecombinant gene encoding L-LDH is a heterologous or exogenous LDH gene.9. The method according to claim 7, wherein said Monascus straincomprises an engineered gene deletion and/or inactivation in a geneencoding an enzyme selected from the group consisting of pyruvatedecarboxylase (pdc), fumarate reductase, alcohol dehydrogenase (adh),acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc),D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (I-ldh) and anycombination of said genes.
 10. The method according to claim 7, whereinsaid Monascus strain comprises at least one engineered gene deletionand/or inactivation in an endogenous gene encoding pyruvatedecarboxylase (pdc), D-lactate dehydrogenase (d-ldh) and/or L-lactatedehydrogenase (I-ldh).
 11. The method according to claim 1, wherein saidorganic acid is succinic acid and/or fumaric acid and which comprisesone or more of the following: a) one or more heterologous or exogenousgenes chosen from phosphoenolpyruvate (PEP) carboxykinase gene, malatedehydrogenase gene, fumarase gene, fumarate reductase gene, isocitratelyase gene and/or malate synthase gene; and/or b) at least oneengineered gene deletion and/or inactivation of an endogenous succinicacid and/or fumaric acid consumption pathway or a gene encoding aproduct involved in an endogenous pathway which produces a metaboliteother than succinic acid and/or fumaric acid.
 12. The method accordingto claim 11, wherein said organic acid is fumaric acid and wherein saidmicro-organism comprises one or more heterologous genes selected from apyruvate decarboxylase gene, a malate dehydrogenase gene, and a fumarasegene.
 13. The method according to claim 11, wherein said micro-organismfurther comprises an engineered gene deletion and/or inactivation in oneor more genes encoding an enzyme selected from the group consisting ofpyruvate decarboxylase (pdc), alcohol dehydrogenase (adh),acetaldehydedehydrogenase, phosphoenolpyruvate carboxylase (ppc),D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (I-ldh),glycerol-3-phosphate dehydrogenase, Isocitrate dehydrogenase and anycombination of said genes.
 14. The method according to claim 11, whereinsaid organic acid is succinic acid and which comprises at least oneengineered gene deletion and/or inactivation in an endogenous geneencoding succinate dehydrogenase or fumarate reductase.
 15. The methodaccording to claim 1, wherein said organic acid is produced at a yieldof at least 0.5 g/L from hexose or pentose sugars or combinations ofhexose and pentose sugars.
 16. The method according to claim 15, whereinsaid yield is at least 2 g/L.
 17. The method according to claim 1,wherein the species within the Monascus genus is Monascus ruber.
 18. Amethod for producing a purified organic acid, which method comprises themethod of claim 1 and further comprises the step of recovering theorganic acid from the cultivation medium.
 19. An isolated micro-organismof the genus Monascus, tolerant to an organic acid concentration of atleast 50 g/L at a pH of less than 5.0, which has been geneticallymodified for increased production of said organic acid.
 20. Themicro-organism of claim 19, which is tolerant to an organic acidconcentration of at least 50 g/L at a pH of less than 3.0.
 21. Themicro-organism according to claim 19, which comprises one or more of thefollowing: a) one or more recombinant genes involved in the productionof said organic acid; and/or b) one or more engineered gene deletionsand/or inactivation of genes involved in an endogenous metabolic pathwaywhich produces a metabolite other than the organic acid of interestand/or wherein the endogenous metabolic pathway consumes the organicacid of interest.